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Real-Time PCR User Guide

1. Fist Tyco Fidi LI Lorient 5 Have Indluded Bl tex E B wc El lll ie El Figini Tei 01 10 Close the Detector Editor 11 Click the Open Mapping File icon F fisMark Real Time PER Anabyzis fie b vem Pen Jh reb PEER West a M Op Run Senay Gare E spent iens VE Fab Cu fl NOTE If you are analyzing a 96 96 chip select M96 Assay SBS96 dsp 48 Fluidigm Real Time PCR Analysis Software User Guide Setting up a Detector Assay Plate Open Detector Mapping File Look in O ReagentPlate M48 Assay SBS9 amp Left dsp M48 Assay SBS96 Right dsp My Recent M96 Assay SBS96 dsp Documents File name My Network Files of type Dispense Mapping files dsp vv Fle Ede Ves Report Toh Hep zi il Che Run Summary B33220011 z w Aniya Views eo Heup Chev Pie ees ES Desa happ ee E ELT A TER a h leig L I o e sie a plate Kr rimi i Name Blanes Name i i 1 i Test Tere Test FI Test fepe bc apan aeos plsbe Tarn i reas uz aiii Numi jams hal bel 1 bel bel Ba we j amp J we Puts erg ie Hane Nara umi O e Man Hare Rai Raf af Barcode ueg Esa O Detector Carteri Probe lupe Timani Eondente Forder Ta 0l Your detector plate setup is complete NOTE You can also copy and paste sample assay names directly from Microsoft Excel spreadsheets Fluidigm Real Ti
2. Fluidigm Real Time PCR Analysis Software User Guide 133 Preparing Sample Pre Mix and Samples 1 Prepare the Sample Pre Mix as shown below Sample Pre Mix Component Volume Volume per Volume for Volume for per Inlet Inlet with 48 48 96 96 pL Overage Dynamic Dynamic pL Array IFC pL Array IFC pL 60 samples 120 samples 2X SsoFast 360 0 EvaGreen Supermix with Low ROX Bio Rad PN 172 5211 20X DNA Binding 0 25 0 3 18 0 36 0 Dye Sample Loading Reagent Fluidigm PN 100 3738 e green cap Total STA and Exo I 2 25 2 1 treated sample C a Table 6 Sample Pre Mix solution 2 Aliquot 3 3 uL of Pre Mix for each sample and add 2 7 uL of STA and Exo l treated sample 3 Vortex the Sample Mix solution for a minimum of 20 seconds and centrifuge for at least 30 seconds Prepared reactions can be stored for short times at 4 C until the samples are ready to be loaded into the chip 134 IMPORTANT Use caution when pipetting the 20X DNA Binding Dye Sample Loading Reagent as bubbles can be introduced Fluidigm Real Time PCR Analysis Software User Guide Preparing the Assay Mix Preparing the Assay Mix 1 Dilute the 100 uM stocks of combined Forward and Reverse Primers for each assay to a final concentration of 5 uM as shown Component Volume per Volume per Inlet Volume for 50 pL Inlet pL with Overage Stock pL pL 100 uM each mixed Forward 0 25 0 3 2 5 and Reverse Primers Ta
3. Unknown R48 CO4 SO Unknown 1 00 D04 Unknown b Drag to any place on the bar Chip Explorer Analysis Views 5 gj Chip Run Gene Expression A Analysis Views B wr Sample Setup m Sample Mapping View Detector Setup EZ Detector Mapping View E E Results Table dal Show Selected Rows Drag 3 column header here to group by that column Experiment Information Chamber Sample FAM MGB ID Name Type rConc Name i Unknown 1 00 DOL Unknown R48 C02 S01 Unknown 1 00 D02 Unknown R48 CO3 501 Unknown 1 00 DOS Unknown R48 CO4 son Unknown 1 00 D04 Unknown Chip Explorer Chip Run Gene Expression GE S Results Table d Show Selected Rows A Analysis Views c QU Sample Setup EN 1 EN 1 1 Sample Mapping View be LAC B Detector Setup m Experiment Information E Detector Mapping View Ec Sample FAM MGB ID Name Type rConc Mame Type gt Name DOL i Name DOZ i Name DOS Name D04 The data are now grouped by name in the Results Table 60 Fluidigm Real Time PCR Analysis Software User Guide Working with Analysis Views 4 Group as many elements as you like by dragging and dropping as in the example below Analysis Views RB Ix Results Table d Show Selected Rows Cm ae pU tq CUT CHENENEEN CC RENE LUN FAM MGB IB EE EE Name EN vee S01 Unknown 1 00 D4 Analysis Views GD Ec Results Table da
4. Using the Animate Feature In the Graph Views watch an animation of each cell on the entire chip in sequence Use this feature while in the Results Table Image View and or Heat Map 1 Clicka cell or row 2 Click Play Watch the Normalized Intensity and Amplification graphs as each cell is displayed in sequence 96 Fluidigm Real Time PCR Analysis Software User Guide Using the Graph View FAM Chamber FAMJMGB ct ID Ll Name Type rConc Name Type Value S A 548 Unknown 1 00 401 Unknown 548 402 548 Unknown 1 00 402 Unknown 548 403 548 Unknown 1 00 403 Unknown 48 A04 4 i Santia N ca SEM n ie moths Click a row in the Results Table in this 548 405 548 Unknown 1 00 405 Unknown d th li k P l 548 406 548 Unknown 1 00 406 Unknown examp e an en CIIC ay 546 A07 548 Unknown 1 00 A07 Unknown The highlighted row is displayed in the 548 408 548 Unkni 1 00 408 Unkni a 1 graph views and then the animation jumps 548 409 548 Unknown 1 00 409 Unknown SAIS eae Tone T aan an to the next cell in sequence and so on until 548 A11 548 Unknown 1 00 A11 Unknown you click Stop or the animation runs 548 412 548 Unknown 1 00 A12 Unknown through the entire chip 548 A13 548 Unknown 1 00 A13 Unknown 548 A14 548 Unknown 1 00 A14 Unknown 548 A15 548 Unknown 1 00 415 Unknown Unknown 1 00 A16 Unknown 548 417 548 Unknown 1 00 4
5. e 94 FOINSE rrr 94 FOGIS LOO Ghee aise hee et de hh cr ael o ea eoe ae dea 94 cChanoina PASS ral eat a8 wet ae ee ena Bhd bow aeeb ae ae Sha we Saad A 95 Using the Animate Feature aaa 96 Selectilig a SINGIS Cell su xm x dence SCHEDE Coe n oe PSP a a 98 Selecting More Than One Cell lllnn 98 Cross Highlighting and Selecting l enne 100 EXDOPEIDIOHD dU a ui aeta viendo Ra et den deci Do de ciere Oe eu ede ee ae p rios 101 Exporting Data from the Results Table 2 000 eee 101 Opening Exported Data csv files en 102 Calculating Delta Ct Sample Values lle 105 Calculating Delta Ct Detector Values eens 109 Deita Delta Ct ValueS ax acne debe Xo aei x eo wa 4 Xe s 110 Fluidigm Real Time PCR Analysis Software User Guide Chapter 4 Chapter 5 Appendix A Viewing Delta Ct Data inthe Heat Map ne Viewing Chip Run Data in the Calibration Curve View INUFOGUCHION usado ded add dhe nb aw gol d hbk dose ok ot dod dub Ae ee And 3 COV IM PAG EX AMOS usu doo acie dece Qo ie a a eased cie CR do eed Using CCVM to Determine Concentration Levels of Unknown Samples Viewing Multiple Calibration Curves enn qPCR Melting Curve Analysis Introduction to qPCR MCA Chip Runs e Running a Chip with a qPCR MCA Protocol llle DSE UEICHSCOMSREROSSOCAELICRZIQOOILIOIDCO Se ead we Viewing the Tm Ranges 0 ee Editin
6. e Keep the amplification room where PCR machines are housed separate from the room in which PCR reactions are assembled DNA free laminar flow hood Using Controls e Include whenever possible a positive control that amplifies weakly but consistently Using a strongly positive control sample may result in excess amplified product which may serve as a source of contamination Use well characterized negative samples such as lambda DNA e Include reagent controls containing all the necessary reagent components but excluding test DNA e Use decontaminating enzymes such as uracil N glycosylase UNG or Uracil DNA Glycosylase UDG to further minimize the likelihood of contamination What You Need for Experiments This section describes the materials that you need to perform your experiments including reagents we support and sample requirements In addition you need the following e BioMark HD System e IFC Controller e 48 48 Dynamic Array IFC or 96 96 Dynamic Array IFC e 20X GE Sample Loading Reagent Fluidigm PN 85000735 store at 49C e 2X Assay Loading Reagent Fluidigm PN 85000736 store at 42C e Deionized DNA free DNase free RNase free water store at room temperature e DNA Suspension Buffer 10 mM Tris pH 8 0 0 1 mM EDTA TEKnova PN T0221 store at room temperature e Sample Mix Prime probes sets Samples of interest 20 Fluidigm Real Time PCR Analysis Software User Guide Supported De
7. errans 162 Required EQUIDMeNE criari esatti end ere Xu eU XR CE x EPA 162 Sotware Requirements eriw arasi Re Bad CARD Re EO Cn ea 163 Cell Sorting Proced re avon ia aan de cio Pus A ek bci AC aw AC Wea doe as 163 Reverse Transcription Specific Target Amplification RT STA 163 Fluidigm Real Time PCR Analysis Software User Guide Preparing LOK ASSAYS a qoc oe eeu oe See ab I Ee ee Oe ERE ES 165 Preparing Sample Pre Mix and Samples 1 aa ee ee 165 Priming and Loading the Dynamic Array IFC 2 0 2 166 Using the Data Collection Software 0 nes 166 lu 6 TUPPPTUIPTTT 167 Fluidigm Real Time PCR Analysis Software User Guide 9 10 Fluidigm Real Time PCR Analysis Software User Guide About this User Guide How to Use This Guide The following chapters provide information about the analysis software and protocols for Real Time PCR on the BioMark or BioMark HD Systems Document Conventions This guide uses specific conventions for presenting information that may require your attention Please refer to the note conventions below CAUTION This convention highlights potential bodily injury or potential equipment damage upon mishandling of the BioMark System Read and follow instructions and or information in a caution note very carefully to avoid any potential hazards WARNING This convention highlights situations that may require your attention May also indicate correct usage of ins
8. p Pass gFal No Call Clear Clear All In Eva Green at 20C Chamber ID Sample Name Detector Name ct Cal Y Tm Detector gt Eva Green at 20 C A01 Ha amp lial EA Ba amp n Ey X Eva Green at 20 C A02 60 a5 Normalized Intensity Amplification Eva Green at 20 C A03 60 25 Eva Green at 20 C A04 60 95 us D Eva Green at 20 C 405 60 95 4 00 i Eva Green at 20 C 406 60 95 0 80 Eva Green at 20 C A07 6o 95 xn Eva Green at 20 C 408 60 25 amp 0 60 3 Min Eva Green at 20 C 409 60 95 0 40 Eva Green at 20 C A10 60 95 nee Fos Craan SE OD C Ati en ac ana 0 20 Graph Viewer Tool Bar Expand collapse pane 3E Threshold ke Show No Call E Log raph L1 Full Range iy Pass ag Fail iy Mo call E Clear ay Clear Al gt Fl Clear or Clear All Auto R Hide No Call UD CS manual changes ke show No Call L Full Range r3 Manual Range Change threshold by clicking Full Range and dragging threshold line Auto Range or Manual Range 92 Fluidigm Real Time PCR Analysis Software User Guide Using the Graph View Toggling the Threshold Click the Threshold button to apply a C threshold line to the amplification graph Amplification With threshold baseline Fluidigm Real Time PCR Analysis Software User Guide 93 Viewing Chip Run Data in the Data Analysis Software Using the Graph Edit Button IMPORTANT The Edit button is
9. 21 971 27 277 21 271 24 274 7 25 275 26 575 27 377 22 975 25 975 35 235 31 911 37 217 31 811 35 935 7 35 935 37 817 Ea 35 535 35 sao 3T 54T 47 547 41 541 44 544 45 545 7 1r 4E 7 37547 E EEEL 55 5 5 51 851 52 587 51 551 51 5547 Lam KELE 57 357 esses 55 585 uonisog au ejdures 7 Optional Click Show Grid again to toggle the grid on and off To change the color of the borders of selected cells 1 Click the Preferences button 2 Click the Selection frame color rectangle The color palette opens 3 Click a color 4 Click OK Frames of selected cells now show the new color in the heat map Fluidigm Real Time PCR Analysis Software User Guide 87 Viewing Chip Run Data in the Data Analysis Software Selected cells in green Show Cell Text To show the details of a cell in text 1 Click Preferences 2 Click Show Cell Text Heat map cells are enlarged and text is now visible Analysis Views Chamber ID 502 402 Sample Mame 502 Detector Mame 402 Ch 13 34 cese dou Call pass l Sample Type Unknown og tf g 3 m B 3 o a Sample Type Unknown Sample Type Unknown Sample Type Unknown Sample Type Unknown Detector Type Test Detector Type Test Detector Type Test Detector Type Test Sample rConc 1 Sample rConc 1 Sample rConc 1 Sample rConc 1 3 Optional Click Show Cell Text again to togg
10. Type Name Ref Detector Setup Type Name yo ae Type Name Ref Click and drag to select Press and hold the CTRL key while clicking individual cells 6 OPTIONAL Click the Detector Plate Map icon F The map opens and shows selected cell s relative to the entire detector plate 46 Fluidigm Real Time PCR Analysis Software User Guide Setting up a Detector Assay Plate Detector Setup J editor m BS ee ee Type Refer Type Refer Type Refer Name testOl Mame testOl Mame testOl Type Refer Type Refer Tyne Refer Name test0i Name testOl Name test01 Type Refer Type Refer Type Refer Mame test i Mame testi Mame testi ee Test Type Type Type Test Type Test Type Test LL LL Alaram LL LL 7 Click Editor The Detector Editor opens 8 Complete the Detector Editor a Select the appropriate type NOTE If you want to identify a reference before moving on see Calculating Delta Ct Detector Values on page 109 I b Enter the Detector Name Detector Editor Detector Editor Type v Type Bi Reference Detector Name Detector Name IT Fest 01 Halen Reference Reference 9 Click Update The Detector Plate Setup reflects the updates Fluidigm Real Time PCR Analysis Software User Guide 47 EEr WE ChpRun Gere Epia TAa uates E Sample Sena Vd Sanple Happ View E oe Sete
11. 13 Detector Inlet Position uonisog eju ejdure s 31 S31 34 934 36 S36 40 S4O 41 S41 42 542 43 543 44 S44 45 545 46 546 42 S42 51 S51 53 S53 54 554 64 S64 70 S70 72 872 73 873 75 875 72 S72 79 379 21 S21 24 584 27 S87 91 591_ CECE The change is reflected in the heat map and in the legend 6 Optional Click Invalid Color Data mm to change the color of failed cells a Click a color square Or b Click Define Custom Color to pick a color other than a basic color 80 Fluidigm Real Time PCR Analysis Software User Guide Using the Heat Map PX Basic colors EI Ge See ee ETE LE 4 ERE Ree EN EEE Ee EE EEE Ee HHENENM NM Custom colors Custom colors 8 8B BUONA SD HERE nm mamanman 2 167 Green 226 tom Colors ColorlSolid Lum 150 Blue 32 Define Cus c Click OK Color Range Pane in the Color Lookup Editor You can change the following parameters e Number of color segments e Minimum value e Maximum value e Auto Range S Color Lookup Editor Color Scheme CtYellowToBlue Data Range Number of color segments Minimum Value Maximum Value 28 50 Changing the Number of Color Segments Change the segments shown in the heat map 1 Type a value 2 minimum 2 Click OK to reflec
12. they also can be used with standard cycling conditions This protocol was thoroughly tested on a wide variety of assays and good results can be expected from the majority of assays For especially difficult assays the cycling conditions can be modified Required Reagents e 2X TaqMan PreAmp Master Mix Applied Biosystems PN 4391128 e 20X TaqMan Gene Expression Assays Applied Biosystems e 2X Assay Loading Reagent Fluidigm PN 85000736 e 2X Master Mix for Fast Cycling Quanta PerfeCTa qPCR Fast Mix low ROX Quanta Biosciences PN 95078 012 or VWR PN 101419 220 or e TaqMan Fast Universal PCR Master Mix Applied Biosystems PN 4352042 or TaqMan GTXpress Master Mix Applied Biosystems PN 4401892 or e TaqMan Fast Advanced Master Mix Applied Biosystems PN 4444557 e 20X GE Sample Loading Reagent Fluidigm PN 85000746 Required Equipment e Standard 96 well Thermal Cycler e IFC Controller MX for the 48 48 Dynamic Array IFC or HX for the 96 96 Dynamic Array IFC e BioMark HD System Required Software Fluidigm Real Time PCR Analysis Software v 3 0 2 or higher and BioMark HD Data Collection Software v 3 0 2 or higher is required for this advanced development protocol 156 Fluidigm Real Time PCR Analysis Software User Guide Specific Target Amplification STA Specific Target Amplification STA STA allows for a multiplexed preamplification by using a pool of gene expression assays as the source of the
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14. 22 04 0 25 0 42 f VicGAPDH Weighted Linear 72 Std2 S05 21 83 0 25 0 31 wv VicGUSB Weighted Linear Std2 506 22 34 0 25 0 00 A VicHPRT Weighted Linear 72 Std 1 EU T VicPGK1 Weighted Linear 72 Std 1 E ig I S03 m Std 6 1516 0 00 10 0 Std6 S17 31 0 00 6 63 WA Std6 518 19 0 00 x Std5 513 0 0039 2 41 f J Record 11 of 13 gt J Mal Record 1 of 72 ecco 3j af p Pass grail Toggle Log VicGUSB 14 15 165 44 3200 Open BST R 0 7489 31 00 30 00 29 00 28 00 27 00 26 00 25 00 i 24 00 a 23 00 22 00 2100 E 0 0001 0 0100 1 0000 Concentration Manually failed points The analysis software can now use the calibration curve data to predict the approximate concentration of unknown sample types 6 Gotothe Analysis Views page Select the Results Table 8 The approximate values are listed in the Calibrated rConc Calibrated relative concentration column J Relative Concentration for unknown samples BioMark Real Time PCR Analysis SEE File Edit View Report Tools Help 1 Ll E e Chip Explorer Analysis WIS 6 Chip Run Summary 1131055065 E Ec Results Table cs Show Selected Rows el 3 gnum View lt lt m Sample Setup H E Detector Setup lity Call Threshold Value Calibrated rConc Quality Call Threshold 0 9
15. 2X reaction reagent into each well of the PCR plate and seal the plate with adhesive film Store the plate on ice if cells are sorted within one day Otherwise store at 20 C 3 Using a FACS instrument sort cells of interest directly into the plate containing the 2X reaction reagent 4 Seal plates with adhesive film vortex for 10 seconds and centrifuge at 1500 RPM for 1 minute 5 Use immediately or store at 80 C NOTE 1 The FACS instrument needs to be carefully calibrated to deposit single cells in the center of each well of the PCR plate 2 Sort in batch mode using a FACS machine 3 Sort cells into the same plate that will be used for thermal cycling Reverse Transcription Specific Target Amplification RT STA This reverse transcription preamplification procedure works for both standard and fast TagMan gene expression applications 1 In a DNA free hood pool all TaqMan Gene Expression Assays and dilute with DNA Suspension Buffer so that each assay is at a final concentration of 0 2X For example pipette 4 uL of each of the 96 TagMan Gene Expression Assays 384 uL total into a 1 5 ml sterile tube and add 16 uL of DNA Suspension Buffer resulting in 400 uL of 0 2X primer probe mix Fluidigm Real Time PCR Analysis Software User Guide 163 2 For each 96 well PCR plate containing sorted cells prepare the reaction mix by combining 300 uL of the 0 2X Primer Probe mix 24 uL of SuperScript III RT Platinum Tag Mix and 1
16. 3 fps v Clear Clear All S Threshold Play Toolbars Show No Call gt Amplification Melting 1 50 024 1 20 0 20 0 90 S 016 ME pt d S 0 60 5 E a ee 0 08 0 30 0 04 SS SSS ee 0 00 0 00 1 11 21 Cycle Temperature I Eva Green at 20 C The Tabbed view has two tabs of graphs for each probe type defined in the chip run qPCR and MCA The qPCR tab shows Normalized Intensity PCR and Amplification Mormalized Intensity Melting 1 80 124 ses 0 20 1 AY 20 ID Ad 6 T ES z 0 90 Rd amp 0 12 0 60 t 0 08 0 30 f 0 04 a NA 0 00 MER 61 71 81 91 61 71 81 91 Temperature Temperature Eva Green at 20 C qPCR Eva Green at 20 C MCA The MCA tab shows graphs for Normalized Intensity MCA and Melting Gl Tabbed 2 amp Threshold Show No Call XA Lo ange i il 4 Stop 3fps v Toolbars Eva Green at 20C Chamber ID Sample Name Detector Name NC 004741 S50194 Ct Call Tm 999 00 Normalized Intensity Amplification Tep 1 50 1 50 130 1 20 E E 0 90 iii 7 oso M Qut 0 60 ES 0 30 n 0 30 0 00 x 1 11 21 1 11 21 Cycle Cycle Eva Green at 20 C gPCR Eva Green at 20 C MCA The Melting Graph The Melting graph displays the T curve for the selected chambers A green vertical line represents a T peak inside the T detection range Melting 0 24 0 20 S 0 16 z amp 0 12 0 08 B LL
17. 866 358 4354 within U S or 1 650 266 6100 outside U S or email techsupport fluidigm com Preparing the Reverse Transcription RT Reaction Assembly 1 For each well of a 96 well plate that will be used for sorting prepare RT mix solution Component 5X VILO Reaction Mix 20U uL SUPERase In 1096 NP40 Nuclease free Water Total Table 1 RT Mix Solution 1 2 Pipette 5 uL of RT Mix Solution 1 into each well to be used of the 96 well PCR plate 3 Sort individual cells or sort up to 10 cells directly into the same plate containing RT Mix Solution 1 L Q NOTE Sort cells into the same 96 well plate that will be used for thermal cycling 4 Seal the plate and vortex thoroughly for 15 seconds Fluidigm Real Time PCR Analysis Software User Guide 143 co Oo Ul Pre chill centrifuge to 42C Centrifuge plate briefly at 49C Immediately freeze the plate on dry Ice Store plate at 809C or thaw plate to use immediately Denaturation of RNA 1 4 144 When you are ready to perform RT cycling a Thaw samples on ice b Use a pre chilled centrifuge maintained at 49C to spin the plate briefly c Preheat thermal cycler to 659C d Transfer the samples to the thermal cycler Condition Denature Temperature 65 9C Time 90 seconds Snap chill the plate on ice immediately for 5 minutes and centrifuge briefly at 4 9C Prepare enough RT Mix Solution 2 for all sorted wells accordi
18. Analysis Views 4 Replace the column headers by dragging them from the Customization dialog to their original position Dropdown Menus on the Column Headers Each column header has a dropdown menu Place your cursor over a header to reveal the symbol Analysis Views 3 H Results Table Drag a column header Experiment Informe Chamber ID E RUI1 C31 1 Click the dropdown menu symbol to display the menu 2 Click a location to go to that location Analysis Views GB Sj Results Table d show _ Drag a column header here to group b Experiment Information Chamber Sample ID Alama gt RO1 CO4 ROC S R01 C01 E RO1 CO5 R01 C06 all RO1 CO7 Custom x Results Table ul show Selected Rows R01 c08 Ea Mon blanks Drag a column header here to group by that column R01 C09 RO1 CO1 E A t Inf ti RD1 C10 R01 C02 nrormation RIDE RD1 CO3 Chamber Sample Poy Co4 ID Name Type RO1 C12 roi cos bk RO1 COS 548 Unknown RO1 C13 ROL COS RO1 C14 RO1 cO 7 RO1 C15 RO1 CO8 RO1 C16 RO1 CO9 RO1 C10 RO1 C17 RO1 C11 RO1 C18 RO1 C12 RO1 C19 ROI CI3 wj RO1 C20 RO1 C21 548 Fluidigm Real Time PCR Analysis Software User Guide 67 Viewing Chip Run Data in the Data Analysis Software 3 Click and drag the menu to size it Analysis Views E Bj Results Table amp lshows Drag a column header here to group b Experiment Information Chamber Sample ID Alame RO
19. C 7 tarot To manually set the corners and analyze the chip 1 Inthe error message click OK 2 Zoom into see the corner cells Fluidigm Real Time PCR Analysis Software User Guide 31 3 Move the red cross hairs to each of the four corner cells Make sure each cross hair is on the outer edges of each corner cell Upper left comer Upper right corner Lower left comer Lower right corner 4 Click Done A NOTE If little or no ROX is present the corner cells are very dark If you cannot see the four corner cells try adjusting the Contrast slider You may have to count the number of rows and columns 48 down 48 across to make sure you are placing cross hairs correctly Find Chamilrer Grid Convers E pl x a 5 ix Ft mo Cond i E By moving the 4 cenam inma select the d comers of tha chip DE Cina Forcing a Manual Corner Find If an automated manual corner find is not satisfactory you can force a manual corner find by pressing CTRL and simultaneously clicking Analyze 32 Fluidigm Real Time PCR Analysis Software User Guide Setting Up a Sample Plate Setting Up a Sample Plate Use this table as a guide when annotating your samples Sample Name Blank Description An unused position Nothing in the chamber NAC d No Amplification Control usually the Taq polymerase is left out of the reaction this is a negative control confirming that positives cannot o
20. Chamber Sample Sample Sample FAM MGE FAM MOE Ci a e e i Name Type rConc Mama Typs Walut Guaity Cal Threshold Sa ant Unikriwn 1 HR Va Babe 92 Pass D Ore vs 14 a AD Unkriwn 1 Test Triar O94 Pass D ore vs Ms SBA Hirira 1 Test TE Ed EST 4 004 Pass ur nj HHAH riknerwn 1 Tiri 1E oS a OM Pais rni AT Sal AG Urikinerwn 1 Tick 16 SCAT 091 Paws D Darts A LAE Uridine 1 Tick 1B 595 3754 05 Pata DUETI End Urknewn 1 Ten 15 5 397 115 08 Pags Ones A cand Mn 1 Tes 16 54511776 034 Pass PI AEG Sa Unkrwn 1 Test LEE u 92 Pass B ore vs z2 d Linirn 1 Tesi 16 eh nda 92 Pass D Ore vs Z3 HEA Unknown 1 Titi 1B 54415875 D Pass Quar iss SHAD Urine 1 Ties 16 SORTS 032 Paws nn 25 548 413 Undine 1 Tiel 15 56137263 D33 Pans D DB 155 25 548 A14 Unkrirun i Tei 15 55415245 055 Pais Outer ss 27 Bagas Urikmewn 1 Tent 16 505208 056 Pars DEn 2 SANG Linien 1 Tes 16 550248 032 Pass PI AEG A Saal Unknown 1 Test 15 612305 OO Pass B Od vs 30 Eua Unkriwn 1 Test 1621556571 955 Pass D Orr vas 31 E4B A18 Unknown 1 Tes 16 mariat 03 Pass Ur nie 30 MBAS Under 1 Tick 1b A71 14666 033 Pans Dog iss 33 BA Uridine 1 Ties 16 ARHI ATA Paws ODE 135 H Sul Ad Urikiri i Ten 18 455619445 085 Pais Q 08r 1555 3 saa Lirimin 1 Tes 16 47757942 Du Pass OG OB 15s 3 maia Unknown 1 Test 16 61 os igi Pass Quer vas AT Salas Unknown 1 Test Lr sz Fx 92 Pass B ore vas 3 40 A28 Unknown 1 Test T5 T5244 05 Pass pu ine d WHEA Likra 1 Teal 16565713 03
21. Control l te ees 40 Using the Dispense Map Editor 0 0 I 40 Setting up a Detector Assay Plate ens 44 ToSetUpthe Detector 2osoxc dues a0ve8 08 0048 behead aes 44 Converting a Chip Run to a More Samples Run 00 0 ee ee ee 50 SEGD di Set UP Samples o yie def Rove Re D EHRCRC al oed Saeed 50 Step 2 SeT UD ASSdVS ido esee dre dioR vane ce eddie She ara 51 Viewing Chip Run Data in the Data Analysis Software Working with Analysis Settings o e 54 Changing the Quality Threshold n 54 Changing the Baseline Correction ln 54 Changing the Ct Threshold Method llle 55 Working with Analysis ViewS ere 57 Using the Results Table QN esee e 57 Using the Image VIEW s suse de CNN COUR REUS acu eR Sol ee IRR 69 Image View TOOL Bal 45 ds sXe cocewoe sre dex da va bu ice 4 71 Adjusting the Size of the Location Reference Map 73 Using Me Reat Mib ax qa AN ce On e ee once e e 3d RC See R3 15 Layout VIGW sasa 3 CNN dos dod y deed ido d XL Oa Ro od 89 Inlet Based VIEW N e he hme re rrr rts 89 ChIDsBased VIEW IN aac addo ox dox e rA ARE E AIRE ak ecd ee ier 89 CUSTOM VIEW AA Mv oe ce wi ty mie a ca Bn aed 90 Usirig ENE GrAQNQAOW doch a die ave cea oe dio 3 Go a eed ee See ees 92 Graph VIAWEF ToolBar ia S a Be BS el DEG Ue DE wale cA RE 92 Togglingthe Threshold e Ie 93 Using the Graph Edit Button
22. Controller 7 Using the IFC controller software run the Load Mix 113x script for the 48 48 Dynamic Array IFC or Load Mix 136x script for the 96 96 Dynamic Array IFC to load the samples and assays into the chip 8 When the Load Mix script has finished remove the loaded chip from the IFC Controller You are now ready for your chip run NM Sample inlets j t Figure 1 48 48 Dynamic Array IFC sample and assay inlets 136 Fluidigm Real Time PCR Analysis Software User Guide Using the Data Collection Software Figure 2 96 96 Dynamic Array IFC sample and assay inlets Using the Data Collection Software 1 o on O0 Ul Double click the Data Collection Software icon on the desktop to launch the software Click Start a New Run Check the status bar to verify that the camera has a green light to indicate that it is ready Remove the blue tape from the back of the chip if this was not done previously Place the chip into the reader Click Load Verify chip barcode and chip type Choose project settings if applicable Click Next Chip Run file a Select New b Browse to a file location for data storage c Click Next 10 Application Reference Probes a Select Application Type Gene Expression b Select Passive Reference ROX c Select Probe Single probe d Select Probe type EvaGreen Fluidigm Real Time PCR Analysis Software User Guide 137 The cycling parameters are given for the two dif
23. ELLE TEETE E FOEDE p em B8 E A a C D 1 File Format BioMark Sample Format V1 0 2 Sample Plate Name Tagged Samples cave to a CSV file 3 Barcode ID 4 Description Tagged Sample Definition 5 Plate Type S8596 6 7 Well Location sample Name sample Concentration sample T 9 A02 M 4 M Tagged Samples evne ya d Ready NL UY 9 Click Save to a CSV file to save the file and select a location to save it Step 2 Set Up Assays 1 Open AssayPlateDefinitionForMoreS Edit the Microsoft Excel file to match your experiment Click Create Plate CSV File A second CSV file tab is added to the file Open the new CSV file tab and double check your annotations Click Save to a CSV file to save the file and to select a convenient location for future retrieval U A W N Fluidigm Real Time PCR Analysis Software User Guide 51 Step 3 Import the Sample and Assay Templates To Import the Sample template files 1 From the Data Analysis software open a chip run you want to annotate Select Sample Setup Click Import under Task Browse to the location where you saved your sample template Click Open Go to File gt Convert to More Samples Chip Run a U A WN To Import the Assay template files 1 From the Data Analysis software open a chip run you want to annotate Select Assay Setup Click Import under Task Browse to the location where you saved your assay template Click Open Go to File gt Convert to More Samples C
24. Guide 39 NOTE If you click an unused cell the Well not used warning appears Using the Replay Control Use the Replay Control to show where and in what sequence the Target Plate receives the samples from the Source Plate Start position End position Clears the map Returns the loading to the start position Advances the loading one row ata Plays the sequence Replay Control time with each click toward the end from start to finish one l row at a time Click it t DE KERU p Moves the loading to the end once to pause jj and position then click again to continue Moves the loading back one row at a time with each click toward the start Using the Dispense Map Editor Use the Dispense Map Editor to record custom load maps for future use After recording your loading sequence you can save it and play it back anytime 1 Click Tools Dispense Map Editor 2 Click New The New Dispense Map window opens 3 Complete the New Dispense Map using the following as a guide 40 Fluidigm Real Time PCR Analysis Software User Guide TE New Dispensing Map RT TST Attributes Name Description mm M Dispensing Source Plate Target Plate Mapping Type Pipette Orientation Columns Tips Tips8 Cancel Wi Using the Dispense Map Editor Unique experiment name or chip barcode Relevant characteristics of the experiment SBS96 or SBS384 48 48 113x 4
25. Guide 83 Viewing Chip Run Data in the Data Analysis Software S Color Lookup Editor Color Scheme CtYellowToBlue Data Range Number of color segments Minimum Value Maximum Value Auto Range i Save 11 86 28 18 3 Click OK to see the changes in the heat map and the heat map legend Saving Changes To save custom parameters that you have set 1 Click Save 2 Enter a name for your custom parameters 3 Click Save Ct Y ellowToBlue Test Cancel The Color Lookup Editor opens 4 Click the Color Scheme menu to see the saved parameters Color Lookup Editor Ct YellowT oBlue_Test Ct YellowT oBlue Ct YellowT oBlue_Test Tm YellowT oBlue S gt Data Range Number of color segments Minimum Value Maximum Value Auto Range Save Location Reference Map 35 25 50 Use the location map to reference your cell of interest within the entire framework of the chip e Click the Location Reference map icon Elto open the map 84 Fluidigm Real Time PCR Analysis Software User Guide Using the Heat Map i Detector Inlet Position tector Inlet Position O O OO Aa NN Map x e m E a E gt T d o e E Legend The legend is a color representation of the C or T values displayed on the heat map e Click the Legend icon m Fluidigm Real Time PCR Analysis Software User Guide 85
26. Image View Overlay 1 Click the Overlay icon to activate the red square grid 2 Click the Overlay icon again to inactivate the red square grid Ana ly sis VIEWS Analysis Views E mage View amp lshow MM S amp 100 ji B 100 m IL Toggle grid off Toggle grid on Contrast Adj ust image contrast Click the Auto Contrast icon Or e Move the contrast sliders by placing your cursor over a slider then click and drag TY E SE I AAARRA RUTQ 700 Contrast l l l l l l l l uA l Light lt gt Dark 74 Fluidigm Real Time PCR Analysis Software User Guide Using the Heat Map Dyes Change the dyes View Image in Each Cycle Use the menu to select an image to view Select number 7 in the menu for example and the image taken at cycle 7 displays in the Image Viewer co Spo cn R oco DR Using the Heat Map The heat map color codes C values for easy reference To access the heat map view 1 Click Analysis View in the Real Time PCR Analysis software 2 Goto Heat Map View Analyziz Views Heat Map View The default heat map opens Fluidigm Real Time PCR Analysis Software User Guide 75 Viewing Chip Run Data in the Data Analysis Software L ligidiem Real Time POR rr lepexk Toas Hole sa m UU TI rest Map Yer M cel Santis Soho ie f vn LAYE Tell bored Viner Detector Setup Detector Inlet Posten Hi
27. Setup Sample Mapping View B Detector Setup Right click a header within a group Click Ungroup to remove the header from the group Analysis Views fH Sj Results Table d Show Selected Rows Name Ei Type Working with Analysis Views Full Expand Experiment Information ES Full Collapse Chamber Sample Sort Ascending Pia ferien a A Sort Descending Name 502 Name 503 E Name S04 3j Name 505 Group By Box Name 506 gA Column Chooser Name 507 ka Best Fit Name S08 M Name 509 Name S10 Best Fit all columns 5 gj Chip Run Gene Expression A Analysis Views m Sample Setup Sample Mapping View B Detector Setup Right click anywhere on the grouping bar Analysis Views fH Ix Results Table d show Selected Row Experiment Information Chamber Sample a Name S01 arrore ID A eee eer Name 503 Name S04 Analysis Views Results Table dal Show Selected Rows gt Full Expand LANE ID 7 ES Full Collapse Experiment Information Chamber Sample ID Name Type Name 533 Name 535 Name 536 Fluidigm Real Time PCR Analysis Software User Guide FAM MGB Name rConc Type 63 Viewing Chip Run Data in the Data Analysis Software Expanding and Collapsing All 1 Right click anywhere on the grouping bar 2 Click Full Expand The grouped windo
28. Views Sample Setup in Detector Setup Task ax Setup Click one of the following New to create a sample plate Export to save a plate for reuse Import to open an existing plate New Export Import Plate Settings Source 96 Wellplate ee Name pet me um Barcode m a Mapping 96 96 Sample SBS96 De Sample Contents Passive ROX Reference Contents References 12 Double click either left or right sample mapping file to determine dispense location NOTE If you are analyzing a 96 96 chip select M96 Sample SBS96 dsp Open Sample Mapping File Look in SamplePlate E M48 Sample SBS9 amp Left Usp 2 M48 Sample SBS94 Right sp My Recent M96 Sample SB596 dsp Documents 2 Desktop Qn My Documents My Computer File name My Network Files of type Dispense Mapping files dsp Your selection is displayed in light blue left or right 38 Fluidigm Real Time PCR Analysis Software User Guide Using the Sample Mapping Viewer File Edit View Report Tools Help fol E X A E e Chip Explorer Sample Setup amp Chip Run Summary 8888220011 Editor Sample Plate Map C3 Analysis Views i E iz Detector Setup Task ax Setup Click one of the following C New to create a sample plate Export to s
29. all by right clicking anywhere on the grouping bar 64 Fluidigm Real Time PCR Analysis Software User Guide Working with Analysis Views 4 Select Full Collapse Experiment Information Chamber Sample FAM MGB ID Mame Type rConc Name 1501 Type Before full collapse zi ID R47 CO03 E ix Results Table d Show Selected Rows ES Full Expand PE rca Be Clear Grouping Experiment Information Sorting Columns 1 Right click a column header ae T Analysis View T E Res ults Table J Show Experiment Information Chamber Sample ID Name Name 533 Name 534 Name 535 After full collapse 2 Choose either Ascending or Descending to sort that column accordingly ij Chip Run Gene Expression A Analysis Views o m Sample Setup iy Sample Mapping View Bil Detector Setup Experiment Information Chamber Sample ID Name p m 4 Sort Ascending iR48 CO1 zi Sort Descending M R48 C02 501 T Clear Sorting R48 CO3 501 _ R48 CO4 501 Group By This Column R48 COS 501 Group By Box E R48 C06 501 Zl Column Chooser a R46 CO ima Best Fit L R48 C 8 R48 CO9 S01 X Clear Filter R48 C10 501 Best Fit all columns R4R C11 nt TT Inner Fluidigm Real Time PC
30. below Click Detector Setup FA BioMark Real Time PCR Analysis B wy Sample Setup is Sample Mapping View Type Detector Name Reference Use F2 key to update and move to the next well Click Test Fluidigm Real Time PCR Analysis Software User Guide 109 Viewing Chip Run Data in the Data Analysis Software Detector Editor k E Type Bi Test Detector Name reference to detector referenc Test e reference to detector reference O1 Detector reference 01 Reference Detector reference 01 v B ef Detector reference 01 Delta Delta C Values The AAC values are available to you after the sample and the detector A C values are calculated see Calculating Delta Ct Detector Values on page 109 and Calculating Delta Ct Sample Values on page 105 When sample and detector AC values have been calculated click Analysis Views to see A AC data F Biaklark Heal Time POR Anabysis ES Detector Happng Ves 8 wf Ti rd H F T wf D DI LIF aT wv Tr PET 0 92 wv Tr 1 0 83 wv Trl iat 0 95 wv c DIT 1 80 0 96 wv c DIT iat i v Tr rl LE v Tr rl EE DI wr aTi H Un Di x ant Um oid wv iih E 3 E ao w zd S m ae x tube Li Dick ha Arakna bettors m xf Cuber 1 3 Ta arite a chai ran ide bor the fret nm e Aire ardor pa meer im changed ose x tube i aa x tuper ee 8 96 ww tube iti Anaka Taing j a x tuper ii Araks a chip sun urg ore cil B C Bus
31. can be used for a variety of applications This specific protocol has been tested for gene expression targeting 1 and 10 cells on the BioMark and BioMark HD System and should serve only as a guideline for any customers interested in qPCR dye experiments We recommend examining melting curve Tm and C for all assays alongside a positive control sample The protocol involves performing Specific Target Amplification STA which enriches samples for loci of interest STA retains relative abundance between loci and permits quantitative C information to be derived See Devonshire et al BMC Genomics 2011 12 118 for more information on STA preamplification Quantitative PCR is then performed in the presence of a DNA binding dye known as EvaGreen dye Quantitative PCR thermal cycling protocols are immediately followed by acquisition of a melting curve Tn to allow assessment of reaction quality See Mao et al BMC Biotechnology 2007 7 76 for further information on the physicochemical properties of EvaGreen dye See Devonshire et al BMC Genomics 2011 12 118 for more information on STA preamplification This two step protocol is optimized for both the BioMark and BioMark HD systems It includes a fast master mix and a relatively short qPCR protocol We have validated a supermix that has EvaGreen and ROX already incorporated The protocol also uses a 2 step VILO CDNA synthesis kit Required Reagents 142 e SuperScript VILO CDN
32. chip run creatingnew 29 open existing 29 chips 48 48 Dynamic Array IFC 17 benefits of 18 features of 18 overview 17 18 contacting Technical Support 3 contamination prevention of 19 controller see IFC Controller 18 controls using in an experiment 20 Ct threshold methods Auto Detectors 56 User Data Detectors 56 User Data Global 56 CtThreshold Method changing 55 Fluidigm Data Collection Software User Guide Data Analysis software changing Baseline Correction methods in 54 changing CtThreshold Method in 55 changing Quality Threshold in 54 experiments what you need for 20 Fluidigm contacting 3 handling nucleic acid 20 IFC Controller overview 18 Linear Derivative Baseline Correction 55 Linear Baseline Correction 55 menu bar in BioMark Dynamic Array Analysis Software 24 nucleic acid handling of 20 Optics description of 16 organizing your work 19 167 PCR dedicated laminar flow hoods for 20 exponential phase of 14 fundamentals of 14 handling reagents for 20 real time 14 TaqMan chemistry used in 15 polymerase chain reaction see PCR 14 preventing contamination 19 probes supported types of 21 Quality Threshold changing 54 reagents detection 21 real time PCR advantages of 14 see also PCR 15 report see Chip Preparation report 28 run see chip run 29 Sample Mix 20 software see BioMark Dynamic Array Software 24 supported detection reagents 21 TaqMan PCR chemistry
33. e ern eio a o ade dE odo dB os 19 Supported Detection Reagents lllle s 21 Additional Probe Types e er 21 PCR Master MIXeS v anche NOR nuc cire entree ied Soule e ue eae DE e 21 Sample Requirement yA ee E e 22 DNA Quality NM 9 a4 402 4 dee eh a e ua de OS OR ODA 2 1 a dS 22 CDNA IDDUt Mal Fe so So aes Ae A ee a a Se ne ee au ee xn 22 CDNA SEO DRE hae Ok St af stica fpe cds We o den dicii ok I Ios ed dio Sen E dias 22 nisis 0 slo wot ELE 22 Using Real Time PCR Analysis Software EAURCMING the SoftWare e ao does P ace RC oO a ER Bee awed MA 24 Menus and ICONS s s ade ait ao do v a EU BOCA do Ro eee atat A ORC oe eee t ARCH 24 TOD MENU Bar s xci took duc acer crede old Ge deu ioo kon Hak oe ds d rcs 24 Secondary Menu Bal s s uia 435 arci eio Or o Seo Ae EC ee fo d BS 25 FIG Sn deae waters P Ro Eae Y eb desea em dee ee ae edes q ids 25 BOG ioco sum Pob A E So eeu Pola d s au d deut dore S qe bul hae ee dicas d 25 d m 26 REDOM racio deck haeo eeu rte ak aca 4 eub qc x A UAR e dire died 26 n n Mr r 28 Creating a New Chip RUN hrs 29 Opening an Existing Chip Run oaaae II 29 Finding Corners Manually 0 0c a 31 Chapter 3 Forcing a Manual Corner Find 0 a a 32 setting Upa Simple Plate x pao doxes ax wed Mae ds eae ea or 33 Using the Sample Mapping Viewer es 39 Using the Replay
34. enabled only when the CT Threshold Method is either Auto Detectors or User Detectors Ct Threshold Method Auto Detectors uta Global Auto Detectors User Global User Detectors Toggle Edit Click Toggle Edit in conjunction with Toggle Threshold enable moving the threshold bar to a new position by clicking and dragging it in the lower graph This can only be done in User Data Global or User Data Detector threshold analysis methods 54938 Aaud 5no blank 1 UU RBZ Analyze 548 409 SH6 Blank 1 00 RB3 Nie duc den chanel Click on the Analyze button to e zem Blank 1 00 RB6 update the results Saf hs S C js m r 1nn v AL E Record s Threshold Edit ke Show No Call 38 Log Graph Full R FAM BHQ Chamber ID Sample Name SH6 Detecto Analysis Settings qPCR Analyze a chip run using one of the Ct threshold methods below If you choose a Manual method you can enter the Normalized Intensity desired Ct threshold s 1 05 0 60 Quality Threshold 0 65 S 0 84 0 40 Baseline Correction Linear v 063 e z 0 20 Ct Threshold Method User Global v 0 42 0 00 Threshold FAM BHG 0 188 1 11 21 at 1 i Cycle FAM BHQ Threshold number C Threshold Method can be only User changes as you re Data Global or User Data Detectors position the threshold for Toggle Edit to be enabled line Toggle Log Grap
35. es 29 Opening an Existing CHG RUN uua sso doe e x EO doe EO B RO REP 29 Finding Corners Manually llle nh 31 Setting Up a Sample Plate 1 aaa 33 Using the Sample Mapping Viewer lem IR 39 Using the Replay Control eee WI eee 40 Using the Dispense Map Editor 0 e e KK n 40 Setting up a Detector Assay Plate lon 44 Converting a Chip Run to a More Samples Run leen 50 Fluidigm Real Time PCR Analysis Software User Guide 23 Launching the Software 1 Double click the Real Time PCR Analysis Software icon on your desktop Chip Explorer Chip Run Summary a EM Summary E fluidigm GENETIC ANALYSIS Welcome to Fluidigm Real Time PCR Analysis Software Start by opening an existing chip run or creating a new chip run Quick Tasks Open a Chip Run Create a New Chip Run Recent Chip Runs C 1131080026 INChipRun bml C 1131103019 ChipRun bml AMoreS ample ChipRun bml C 5 41131103019 ChipRun bml Menus and Icons Top Menu Bar View Report Tools File Edit view Report Tools Help New Ctr N Copy Ctrl C New From Current Chip Run i m Pass Ctrl G al Open Ctrl O Fail Ctrl F E Save Ctrl 5 Clear Ctri L Convert To More Sample Chip Run Clear Al Ctrl A View Close Go To gt F1 Export v Chip Explorer v Task C Documents and Settings lab My Documents Fang 136147
36. gt FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB FAM MGB E M MITP DDI _ Initialize with Auto Thresholds W FBX032 0 1 GDF11 01 Trim63 oil ACTB 0 1 CTSB 0 1 FoxM1 0 1 KRT18 0 1 PREP 0 1 VIM 0 1 GAPDH 0 1 TNFRSF10B 0 1 PP1A 0 1 HPRT1 0 1 B2M 0 1 PGK1 oik D Saving or Loading Threshold Settings 56 To save your detector threshold settings for use with another chip run right click anywhere in the threshold table and select Save Table Name the file and choose the location where you want to save the file Fluidigm Real Time PCR Analysis Software User Guide Working with Analysis Views Thresholds _ Initialize with Auto Detectors Thresholds FAM MG Right click anywhere in table FAM MGE Ha Save Table to open menu FAM MGB Apply to all detectors FAM MGB FAM MGE FAM MGB FAM MGB FAM MGE ti Load Table To load your saved detector threshold settings select Load Table Browse to the location of the settings file you want Working with Analysis Views There are three different views Results Table view results in one table e Image View view images from individual cycles Heat Map View view color coded C values Click the Expand Collapse button to display any view at full size or as sp
37. minimum amount of dilution that should be used is 5 fold but if the C also known as C values are consistently below 6 for some of the assays this may need to be increased to 10 fold or 20 fold Use low EDTA TE or DNA Suspension Buffer TEKnova PN T0221 to dilute the products as shown below Table 1 Dilution Table 5 fold dilution 10 fold dilution 20 fold dilution 54 0 uL 129 0 uL 279 0 uL Table 6 Dilution Table 4 Store diluted STA products at 20 C or use immediately for on chip PCR NOTE For larger volume STA reactions adjust the amounts of materials proportionally Fluidigm Real Time PCR Analysis Software User Guide 147 Preparing the Sample Pre Mix and Samples We recommend calculating overages when preparing the Sample Pre Mix solution The volumes in the table below apply to a Fluidigm 48 48 Dynamic Array IFC and a Fluidigm 96 96 Dynamic Array IFC 1 Combine the following to make the Sample Pre Mix solution Table 2 Sample Pre Mix solutions Volume Volume for per Volume per 48 48 Dynamic uL Overage pL Component 60 samples 2X Sso Fast EvaGreen Supermix With Low ROX Bio Rad Laboratories PN 172 5211 Volume for 96 96 Dynamic Array IFC pL 120 samples 20X DNA Binding Dye Sample Loading Reagent Fluidigm PN 100 3738 e green cap STA and Exo I treated sample Total Sample Loading Reagent as bubbles can be introduced IMPORTANT Use caution when pipettin
38. pending patents in the U S and other countries Some Fluidigm IFC Controllers and associated IFCs may be licensed under Caliper Life Sciences V5 6 No right to modify copy use or distribute Fluidigm software is provided except in conjunction with the instrument delivered hereunder and only by the end user receiving such instrument in cases Where the software and the associated instrument are beta test systems NO WARRANTIES ARE PROVIDED EXPRESSED OR IMPLIED ALL WARRANTIES INCLUDING THE IMPLIED WARRANTIES OF FITNESS FOR PURPOSE MERCHANTABILITY AND NON INFRINGMENT ARE EXPRESSLY DISCLAIMED By continuing the installation process user agrees to these terms Please refer to the full text of the software license agreement supplied with the installation media for this application Every effort has been made to avoid errors in the text diagrams illustrations figures and screen captures However Fluidigm assumes no responsibility for any errors that may appear in this publication It is Fluidigm s policy to improve products as new techniques and components become available Therefore Fluidigm reserves the right to change specifications at any time Information in this manual is subject to change without notice Fluidigm assumes no responsibility for any errors or omissions In no event shall Fluidigm be liable for any damages in connection with or arising from the use of this manual For Research Use Only Not for use in diagnostic procedures C
39. primers By using the same assays in the preamplification reaction as the real time PCR reaction only targets of interest are amplified Refer to the Fluidigm Gene Expression Specific Target Amplification Quick Reference PN 68000133 for more information about performing STA Preparing 10X Assays 1 Ina DNA free hood prepare aliquots of 10X assays using volumes in the table below scale up appropriately for multiple runs Volume per Volume per Inlet Volume per Component Inlet pL with Overage uL 50 pL Stock 20X TagMan Gene Expression 2 3 Assay Applied BioSystems 2X Assay Loading Reagent 2 5 3 0 25 Fluidigm PN 85000736 Final Concentration at 10X Primers 9 uM Probe 2 5 uM Fluidigm Real Time PCR Analysis Software User Guide 157 Preparing Sample Pre Mix and Samples 1 Prepare a Sample Pre Mix solution containing the Master Mix and 20X GE Sample Loading Reagent sufficient for the number and type of chips to be run The following table provides the component amounts for one 48 48 or one 96 96 chip Sample Pre Mix Sample Pre Mix Volume per Inlet with for 48 48 uL for 96 96 pL Overage pL 60 for ease of 120 for ease of pipetting pipetting 20X GE Sample Loading Reagent Fluidigm PN 85000746 Quanta PerfeCTa qPCR Fast Mix low ROX Quanta BioSciences PN 95078 012 or VWR PN 101419 220 or TaqMan Fast Universal PCR Master Mix Applied Biosystems PN 4352042 or TagMan GTX
40. the Real Time PCR Analysis Parameters 15 Click Start Run The cycling parameters are given for the two different chip types BioMark HD BioMark Hea re is poni Ramp Rate Ramp Rate Thermal Mix 60 30 5 5 2 2 Hot Start 95 60 5 5 2 3 PCR 30 96 5 5 5 2 Cycles 60 20 5 5 2 4 Melting 60 3 1 1 Curve 60 95 1 C 3s 1 2C 3 s 1 Hot Start 95 60 5 5 2 2 PCR 30 96 5 5 5 2 Cycles 60 20 5 5 2 3 Melting 60 3 1 1 Curve 60 95 1 C 3s 1 2C 3 s Using the Real Time PCR Analysis Parameters 1 Double click the Real Time PCR Analysis software icon on the desktop to launch the software Click Open Chip Run Double click a ChipRun bml file to open it in the software Enter detector and sample information Select Analysis Views We recommend using the AutoGlobal method to set the threshold 6 We recommend using Linear Derivative as the baseline correction method For more information about baseline correction methods contact Fluidigm Technical Support 7 Always compare the T of the intended products to a positive control sample ul Aa W N Fluidigm Real Time PCR Analysis Software User Guide 153 154 8 Click Analyze i NOTE For more information about melting curve analysis see the Fluidigm Real Time PCR Analysis Software v 3 0 User Guide PN 68000088 Fluidigm Real Time PCR Analysis Software User Guide Fast Gene Expression Analysis Using TaqMan Gene Ex
41. the cells to use as a reference e Click the upper left corner to select all the cells e Click and hold while dragging your cursor through cells Fluidigm Real Time PCR Analysis Software User Guide Setting Up a Sample Plate e Click individual cells while pressing the CTRL key Sample Setup Type Name rConc Ref Unknown Unknown Mame rConc UE Unknown 4 OPTIONAL Sample Setup Fluidigm Real Time PCR Analysis Software User Guide Unknown Unknown Unknown Unknown Unknown Type Name rConc Ref Unknown Unknown Click the upper left corner to select all cells Unknown Click and drag to select Unknown Am Unknown Type Unknown 2 Name rConc Ref Unknown Unknown Press and hold CTRL while clicking individual cells Click the Sample Plate Map icon jm The map shows selected cell s relative to the entire sample plate Unknown Unknown Name rConc Ref Unknown 35 36 5 Click Editor The Sample Editor opens 6 Select the appropriate type NOTE To identify a reference see Calculating Delta Ct Sample Values on page 105 Sample Editor Sample N ET Relative Cone NTC Qi Unknown Reference Qi Reference Qi Standard Use F2 key to updat nest well 7 Enter the sample name 8 Enter the relative concentration 9 Click Update The Sample Plate Setup reflects the updates Flu
42. the right pane The selected experiments are plotted on the graph area below 4 Select a row in the Calibrator Table and the corresponding data point in the calibration curve becomes larger Conversely you can lasso or click on a data point in the chart and the corresponding row in the Calibration Table is highlighted NOTE You can lasso a point by pressing the left mouse button and dragging the mouse around the data point s to create a circle Only calibrators with valid C values are plotted in the calibration curve Invalid C values are listed as 999 Calibrators that are auto or manual passed are plotted as blue dots They are considered valid calibrators Calibrators that are manually failed or passed are plotted as red dots and are considered invalid calibrators CCVM only uses blue data points to create calibration curves If there are no valid calibrators no calibration curve is drawn Fluidigm Real Time PCR Analysis Software User Guide 117 Viewing Chip Run Data in the Calibration Curve View 5 You can modify the calls by manually changing the calibrators calls to Pass or Fail via the secondary view tool bar a Select a calibrator you wish to change you can select the row in the Calibrator Table or lasso a data point on the chart File Edit View Report Tools Help Lal gt
43. tubes and gloves for all manipulations involving nucleic acid samples which never leave the DNA dedicated laminar flow hood Change gloves frequently Use aerosol resistant disposable pipette tips Discard tips after each use Use disposable UV irradiated plastic ware Ensure that all equipment including paper pens and lab coats are dedicated for use only in a particular laboratory For example dedicated laboratory coats for each of the PCR rooms Do not bring contaminated workbooks into clean PCR areas Aliquot PCR reagents Wipe PCR hoods daily with DNAZap Ambion or a similar DNA decontaminate Use ultra violet radiation to complete decontamination Ensure that only authorized users work in PCR areas and handle PCR equipment Fluidigm Real Time PCR Analysis Software User Guide 19 BioMark System Prepare reagents in a dedicated DNA free laminar flow hood DNA free areas prohibit any biological material including DNA or RNA extracts and PCR products Also in the DNA free area prepare and aliquot reagent stocks and reaction mixes Handling Nucleic Acid PCR Mixes and PCR Reactions Prevent carry over of amplified DNA sequences by setting up PCR reactions in a dedicated laminar flow hood while keeping post PCR manipulations separate e Add extracted DNA to the PCR reaction mixes in the DNA dedicated Sample laminar flow hood Be sure to prepare the PCR reaction mixes In the DNA free laminar flow hood
44. way Xr eee ao eo BENE n 16 The BioMark HD System Components dee eee ee eee 17 Dynamic Array IFC Components naaa a a 17 48 48 Dynamic Array IFC for Real Time Quantitative PCR 17 96 96 Dynamic Array IFC for Real Time Quantitative PCR 18 BioMark HD System Process Overview rns 19 Before You Begin oL QR rr hrs 19 Supported Detection Reagents llle 21 Additional Probe Types Mee m II 21 PCR Master MIXES oc iva oe RM sor 3 xk EXEC ER ARRA E 21 Sample Requirements uaa a Ss e454 84 Kod eee eee OS HSC eS ES 22 DNA Quality usw RD oa wee ert o eee ee ee oe eR OC 22 aB IMEUE 232299 2 2 9 49 2 99 3 9304 2 45 9 9 1 32 9 48 9 E p po alex 22 CDNA OE DER LEO uq acad go y Edo R1 3 3 3 4 93 2 e EARS EOE CR TD 22 Fluidigm Real Time PCR Analysis Software User Guide 13 BioMark System Real Time qPCR Real time quantitative PCR qPCR is a powerful technique for quantifying changes In gene expression by producing millions of copies of specific targeted regions of complementary DNA cDNA that has been reverse transcribed from messenger RNA mRNA Advantages of Real Time qPCR Historically qPCR has been a time consuming process because of the time required to get gel based end point measured plateau phase results These results tended to be less accurate and did not have as wide a dynamic range as real time PCR With the advent of quantitative data collection during the exponent
45. z x Chip Explorer n X Calibration View amp Chip Run Summary 1131055065 F FAM MGB Show Selected Rows GE Analysis Views ete EE Calibration View F w Sample Setup Name Style Calibrator Count Sin Chick Co Error Call 5 HH Detector Setup FamGAPDH Weighted Linear 54 op Std6 516 29 73 0 00 279 A FamGUSB Weighted Linear 72 Std6 S17 29 11 0 00 1 09 A 3 FamHPRT Weighted Linear 54 Std 6 518 28 59 0 00 ae 0 28 wv ZZ FamPGK1 Weighted Linear L 54 Std5 Si3 25 54 0 0039 1 81 Y FYGAPDH Weighted Linear 72 Std 5 14 26 04 0 0039 0 71 v FYGUSB Weighted Linear 72 F t J FVHPRT Weighted Linear 72 sid zu rand p 0039 2 17 FYPGK1 weighted Linear 72 Std 510 2378 0 0156 3 72 NRC Weighted Linear cq Std4 Sil 24 34 0 0156 1 87 WA VicGAPDH Weighted Linear 72 Std 4 9t2 24 59 0 0156 1 24 A b VicGLISB Weighted Linear Std 3 507 22 86 D 065 2 66 w Task 1X VicHPRT Weighted Linear 72 Std3 S08 23 63 0 065 0 75 A de T Analyze VicPGK1 Weighted Linear 72 Std3 509 23 88 0 065 0 38 Y Click the Analyze button Std2 S04 22 64 0 25 0 51 v To analyze a chip run file for the first time or T EN pene D p After analysis parameter is changed J Record 11 of 13 C Mallal a Record 1 of 72 opp inis T gp Pass g Fail ZA Toggle Log a Analysis Setti VicGUS
46. 0 Trac ru uE Record 12 of 9216 m om T a Theeshold bo Show Wa Cal E Loo Greeh TIAA Range r pass ighel roca MGE Chamber D sSampbNsme Gixpress Detector Namo ct Heeredoed intera t noy Serge oun Leve o chup vun using one of the Co Pedi vef b ya F you choote a Manos metal you da entes the demedti fredde Quality T hemihiotd Baretre Crawctan D ThesiheldNetot Ado feo w Changing the Quality Threshold To change the quality threshold 1 Click Analysis Views 2 Under Analysis Settings enter a different value in the Quality Threshold field Changing the Baseline Correction To change the baseline correction 54 Fluidigm Real Time PCR Analysis Software User Guide Working with Analysis Settings 1 Click Analysis Views 2 Under Analysis Settings select a different option in the Baseline Correction field Linear default Produces higher C values when the amplification is low Linear baseline correction eliminates baseline drift by flattening the baseline Linear Derivative An additional method of baseline correction with a more robust handling of nonlinear baselines and their impact on C estimates The difference between Constant and Linear baseline corrections is shown here Constant baseline correction I i I LJ jl 4 TOU 1s 1B 5195022 725 280 315 Ss Cycle Baselines are rising in the Co
47. 0 v 0 030 0 3422 0 68 v 0 0 76 v 0 030 1 2795 0 68 v 0 0 76 v 0 030 1 3087 0 67 v 0 0 75 v 0 030 1 4854 0 69 v D 0 73 v 0 030 1 4985 0 64 x 0 0 94 v 0 030 i 1 8798 0 82 v 0 0 88 v 0 030 2 1936 0 82 v D 0 90 v 0 030 2 0618 0 83 v D 0 90 v 0 030 2 0862 0 79 v 0 0 75 v 0 030 1 4934 0 67 v D 0 78 v 0 030 i 1 6259 0 68 v D Task ax 0 77 v 0 030 1 5871 0 70 v 0 Analyze 0 76 v 0 030 1 5830 0 67 v D de wae ne wey for the first time or X e Bid rma 2 X 2 After analysis parameter is changed 0 77 v 0 030 i 2 2144 0 89 v 0 0 00 x 0 030 i 1 1871 0 72 v 0 0 00 x 0 030 0 6983 0 42 x 0 Analysis Setting 0 00 x 0 030 2 2487 0 78 v 0 Analyze a chip run using one of the Ct threshold TIKK Record 256 of 2304 gt gt gt a lt i x rasthada halen IF nan abhaaaa a hd iin matha 120 Fluidigm Real Time PCR Analysis Software User Guide Viewing Multiple Calibration Curves You can also view multiple calibration curves at once on the CCVM page To select multiple rows of assay in the Detector Table Ctrl left mouse click the rows of interest Individual curves are rendered in the Graph Area Note however that the Calibrator Table behaves differently when multiple rows are selected in the Detector Table The Calibrator Table is masked gray and the user cannot perform any actions on it This is because the Calibrator Table was designed to only show calibrators for one detector at a time WD CheRun Seep TID
48. 01 Fi Best Ft 1 00 07 m R48 COB8 so 1 00 D08 R48 C09 501 Zk Clear Fite 1 00 D09 R48 C10 i 501 Best Fit all columns i 00 D10 R48 C11 S01 unknown 1 1 00 D1 R48 CI2 SOL Unknown 1 00 D12 R48 C13 501 Unknown 1 00 D13 The grouping bar appears File Edt View Report Tools Help Chip Explorer q X f Analysis Views igi Chip Run Gene Expression fe Results Table Show Selected Rows A Analysis Views Sample Setup column header here to group by that column is Sample Mapping View B Detector Setup Experiment Information Cham Sample FAM MGB ID Name Type rConc Name Type FAM MGB ct Value Quality Call Fluidigm Real Time PCR Analysis Software User Guide 59 Viewing Chip Run Data in the Data Analysis Software 3 Click the column header that you want to group and while holding down the mouse button drag it to the bar as shown below a Click and hold mouse button on header Chip Explorer Analysis Views Ei i Chip Run Gene Expression 3 ix Results Table dal Show Selected Rows A Analysis Views B ur Sample Setup Sample Mapping View x SP a column header here to group by that column S Detector Setup Experiment Information D Detector Mapping View Chamber Sample I7 T NEST ID Name Type rConc Name Type E Unknown 1 00 DO1 Unknown R48 C02 S01 Unknown 1 00 D02 Unknown R48 CO3 501 Unknown 1 00 D03
49. 1 CO1 RO1 C31 RO1 C32 AID RO1 C33 Custom RO1 C34 Blanks RO1 C35 Non blanks RO1 CO1 R 1 C37 RO1 CO3 R01 C38 RO1 C04 RO1 C39 PALA R01 C06 RO1 C40 RO1 CO7 RO1 C41 Ro1 cos RO1 C42 RO1 CO9 RO1 C43 ROL C10 R01 C11 RO1 C44 RD1 C12 R 1 C45 RO1 C13 b RO1 C46 RO1 C14 i RO1 C47 R 1 C48 548 Custom Filters Use filters to narrow your search for a particular parameter In the following example we isolate C quality values below 0 8 1 Click the Quality header menu 2 Click Custom Analysis Views GB Ec Results Table gal Show Selected Rows Experiment Information FAM MGB Chamber Sample FAM MGB ct ID Name Type rConc Name Type Value Quality 9 cau b 548 A01 Unknown 1 0000 detector test 1 NRC D s al 548 A02 Unknown 1 0000 detector test 1 NRC 0 Blanks 548 A03 Unknown 1 0000 detector test 1 NRC i D Non blanks 548 A04 Unknown 1 0000 Test i p c 0 00 548 A05 Unknown 1 0000 Test 0 9 n a 548 A06 Unknown 1 0000 Test l 0 99 39 548 407 Unknown N 1 0000 detector test 1 NRC 548 A08 Unknown 1 0000 detector test 1 NRC 548 A09 Unknown 1 0000 detector test 1 NRC 548 A10 Unknown 1 0000 Test 548 A11 Unknown 1 0000 Test 548 A12 Unknown 1 0000 Test 548 A13 WW Unknown 1 0000 Test 548 A14 E Unknown 1 0000 Test 548 A15 Unknown 1 0000 Test 548 A16 Unknown 1 0000 Test 548 A17 Unknown 1 0000 Test The Custom AutoFilter dialog box opens 3 Delimit your search a Select a f
50. 15 Technical Support contacting 3 UNG using 20 168 Fluidigm Data Collection Software User Guide World Headquarters 7000 Shoreline Court Suite 100 South San Francisco CA 94080 USA Fluidigm Europe BV Luna Arena Herikerbergweg 238 1101 CM Amsterdam Zuidoost The Netherlands Tel 431 0 20 578 88 53 Fax 31 0 20 203 11 11 Fluidigm France Sarl Les Conqu rants Bat Kilimandj aro 1 avenue de l Atlantique 91940 Les Ulis Tel 433 0 1 60 92 42 40 Fax 33 0 160921131 Fluidigm Singapore Pte Ltd Block 1026 707 3532 Tai Seng Avenue Singapore 534413 Singapore Tel 465 6858 7316 Fax 31 0 20 203 1111 Fluidigm J apan KK Luminous 2nd Floor 15 19 Nihonbashi Kodenmacho Chuo ku Tokyo 103 0001 J apan Tel 481 3 3662 2150 Fax 481 3 3662 2154 Fluidigm Shanghai Instrument Technology Co Ltd Room 2907 B Building Far East International Plaza No 317 XianXia Road 200051 Shanghai China Tel 486 21 3255 8368 Fax 496 21 3255 8369 Technical Support send email to TechSupport fluidigm com Phone in United States 1 866 FLUIDLINE 1 866 358 4354 Outside the United States 650 266 6100 On the Internet www fluidigm com support Visit our website at www fluidigm com PN 68000088 C1
51. 17 Unknown 548 418 548 Unknown 1 00 418 Unknown 548 419 548 Unknown 1 00 419 Unknown S4R A f s4a 1 1 anlan 1 Inlenieiai M4 Record 1 of 2304 DO Hormalized ittensiby Amgplifization 0 84 ar 40 07D O d 0 30 Li fa I 2 0456 i 4 020 od I aan 0 28 Fi Ed _ _ gt 15 2 38 b 3 1 amp 11 18 F Cycle Cycle 1 B amp 11 5 33 35 Each cell s data are displayed in the graphs during the animation 3 Optional Adjust the animation speed ao 9 Click aie Choose a viewing speed 4 Click Stop to stop the animation 5 Click Play to continue the animation Fluidigm Real Time PCR Analysis Software User Guide 97 Viewing Chip Run Data in the Data Analysis Software Selecting a Single Cell In the Results Table click a cell to activate its data in the graphs and on the Information bar eese ky ipa eyes In the Image View when the cursor becomes crosshairs click the cell to activate the data in the graphs and on the Information bar R84 44 5060 6050 6 64 4 4 eas 43 7 40 1 28 2 M BH Ww Y x yc 043 85 5 d A oH X 9 oF yia E In the Heat Map click a cell to activate its data in the graphs and on the Information bar Selecting More
52. 3 Pais O08 135 DS A Uridine 1 Tiel ih seb 3t O85 Paws OO iss di A Uridine 1 Tick 16a 1305 AT Paris OO Tas 42 Sob Ad Urikiri i Tes 16 44527045 053 Pais Ones 3 Sale Lirimin 1 Test LE i O94 Papa OQ OB 15s Sad ax Unknown 1 Test 16 rods 95 Pass OG Orr vs Sad ut Unikriwn 1 Test T5 Gas T 91 Pass oer vs dB SAN Unknown 1 Test IEE 05 Pass D Dor 1s l a r 1 iriki 1 Test Ti ses EE O94 Pass B Ode vas iriki 1 Test 17 Soe O92 Pass Oe vas Unknown 1 Test 17 839821 31 093 Pass D Vas Unkn wn 1 Tii 17 841222 OM Pais nid prik riram 1 Tiit 17 8084B12 033 Pari O Dar vas rik riram 1 Teel 17 85 DS 033 Pari OD vas Unknewn 1 Tent 17 95 p 054 Pags O OB ee Lnknawn 1 Test 1 ET 0 45 Fad aor ras irikirik 1 Test 1AA er Fal min vas 4 Lirimin 1 Test Maa OSes Fail Oe vas d Unknown 1 Test SITS 0350602 Fail Onis a Unkn wn 1 Tii WNA 0 139223 Fail Qi prik riram 1 Tiit TBpAZOSGTE 0 SHEA Fail CATIE Unkrirwn 1 Tent 17 Fas 095 Paws O DHT Unknewn 1 Tet 17 ESE 096 Pasa GLOBE rs Vnknawn 1 Test UE 095 Pass QE Unikrarwn 1 Test 17 577 44254 87 Pass Coe vas Mriki 1 Test 17 Se a dr 55 Pass B ire vas Unknown 1 Test I7 S5367 Pasa OOE rik rirani 1 Tiit 17 7 O35 Pir OTS prik riram 1 Tiit 17 7ATEEZ3Z 0 32 Pari Daris Unkrrwn 1 Test 17 52454516 ASE Pass fers Unknawn 1 Test 17 CER 095 Pars a Orr vas Unikraywn 1 Test 17 SEE 85 Pass B dre ves Unknown 1 Test eames OM Pass OW Unknown 1 Test IESS 055 Pass CLOT ESS Unknown 1 Te
53. 44 uL of PCR certified water into a 1 5 ml sterile tube Vortex for 10 seconds Volume per 96 well Plate Component Volume per Well pL with Overage pL 0 2X Primer Probe Mix 2 5 300 0 SuperScript Ill RT Platinum 0 2 24 0 Taq Mix PCR certified water 1 2 144 0 Total 3 9 468 0 Table 1 Reaction Mix 3 To each tube of an 8 well PCR strip add 50 uL of the reaction mix Transfer 3 9 uL of the reaction mix to each well of the PCR plate containing cells from the strip using an 8 channel pipette seal vortex for 10 seconds and centrifuge at 1500 RPM for 1 minute Place the plate onto a 96 well thermal cycler and proceed to RT STA using the thermal cycling protocol below STA 18 Cycles La Taq Activation Annealing Extension Table 2 Thermal cycle conditions 4 Dilute the resulting cDNA product 1 5 with DNA Suspension Buffer Fluidigm Real Time PCR Analysis Software User Guide Preparing 10X Assays Preparing 10X Assays 1 InaDNA free hood prepare aliquots of 10X assays using volumes in the table below scale up appropriately for multiple runs Volume per Volume per Inlet Volume per Component Inlet pL with Overage uL 50 pL Stock 20X TaqMan Gene Expression Assay 2 5 3 0 25 0 Applied BioSystems 2X Assay Loading Reagent 2 5 3 0 25 0 Total Volume 5 0 6 0 50 0 Final Concentration at 10X Primers 9 uM Probe 2 5 uM Table 3 Assay preparation Preparing Sample
54. 48 uL 1X DNA Suspension Buffer 152 uL ma 7 m 0 Table 3 Preparation of 500 nM 10X pooled STA Primer Mix Fluidigm Real Time PCR Analysis Software User Guide 145 Preparing STA Reaction Mix 1 For each well of a 96 well PCR plate that was used for sorting prepare the following mix Per 9 pL 48 Samples with 96 Samples with TagMan PreAmp Master Mix 7 5 Invitrogen PN 4391128 10X STA Primer Mix 500 nM 1 5 0 5M EDTA pH 8 0 Invitrogen PN AM9260G Total Volume Table 4 STA Reaction Mix 2 Aliquot 9 uL of the STA reaction mix to each of the first strand cDNA samples STA Cycling 1 Follow the thermal cycling conditions below on a standard thermal cycler Enzvie 20 Cycles 1 10 cells Senet Activation Exonuclease Exo I Treatment Method For best results we recommend using a cleanup step to remove unincorporated primers This can be done with Exonuclease E coli 1 Just before use dilute the Exonuclease to 4U uL as shown 146 Fluidigm Real Time PCR Analysis Software User Guide Exonuclease Exo Treatment Method Component Per 15 pL Sample 48 Samples with 96 Samples with uL Overage pL Overage pL 4 2 Water Exonuclease Reaction Buffer 10X Table 5 Exo 1 Reaction Solution 2 Add 6 uL of diluted Exo at 4 U uL to each 15 uL STA reaction vortex centrifuge and place in a thermal cycler 3 Dilute the final products to an appropriate concentration for testing The
55. 6 Dynamic Array IFC see Fluidigm 96 96 Real Time PCR Workflow Quick Reference PN 68000130 Using the Data Collection Software The protocols used for data collection are fast protocols 48 48 Select GE 48X48 Fast v1 pcl in the GE folder This protocol takes approximately 26 minutes This cycling protocol is described below Amplification 35 Cycles Taq Activation Denaturation Annealing Extension Temperature 95 C 96 C 60 C Time 1 min 5 sec 20 sec Table 5 48 48 protocol e 96 96 Select GE 96X96 Fast v1 pcl in the GE folder The cycling protocol portion of this is the same as for the 48 48 but also includes the Thermal Mix protocol for the 96 96 Dynamic Array IFC The total program runs approximately 66 minutes The complete program is described below Amplification 35 Cycles Thermal Mix Taq eT Annealing Activation enaturation Extension Temperature 70 C 25 C 95 C 96 C 60 C Time 30 min 10 min 1 min 5 sec 20 sec Table 6 96 96 protocol 166 Fluidigm Real Time PCR Analysis Software User Guide Analysis Views see BioMark Dynamic Array Analysis Software 30 Baseline Correction changing 54 Linear 55 Linear Derivative 55 before you begin handling reagents 20 organizing your work 19 preventing contamination 19 using controls 20 BioMark Dynamic Array Analysis Software launching 24 menu bar 24 BioMark System components of 17 overview 16
56. 6176 ChipRun bml Exit BioMark Real Time PCR Analysis File Edit View Report Tools Help Dispense Map Editor Options Chip Preparation Report Install Test Report 24 Fluidigm Real Time PCR Analysis Software User Guide Menus and Icons Secondary Menu Bar IFluidigm Real Time PCR Analysis File Edit view Report Tools Help C e bi Owls E ij Help New chip Open Save Backward Undo redo Export run chip run chip run forward call CVS navigation file File The File menu has the following options New Open the Chip Run Setup Wizard Open Open location of bml chip run data files Save Save your current run data file with any changes Convert to More Samples Chip Run Convert your chip run to a more samples chip run Close Close your current run data file Export Export Results table data or Heat Map data as csv text file oe Open the location of recently viewed used bml files Exit Close the application Edit The Edit menu options depend on the active window If the Active Window Your Options Are iS ae Sample Setup Undo Ctrl Z Redo Ctrl Clear Sample Setup Ctri K Copy Sample Setup Copy Ctrl C Paste Ctrl Detector Setup mS Undo Ctrl Z Redo Ctrl Clear Detector Setup Ctrl K Copy Detector Setup Copy Ctrl C Paste Ctrl Fluidigm Real Time PCR Analysis Software User Guide 25 If
57. 8 48cs 132x or 96 96 136x Assay Detector Inlets or Sample Inlets Columns or Rows Tips1 Tips2 Tips4 Tips6 or Tips8 4 Click OK to open the new dispense map in the Dispense Map Editor Source Plate Graphical representation of the plate from which the samples and or detectors are pipetted x Dispense Map Editor File Help TT T Y Attributes Source Plate 58596 a Name new Description Dispensing Source Plate 58596 96 36 136x Target Plate Mapping Type Assay Detector Inlets Pipette Orientation Columns Tips TipsB E Recording Control Playback Control BOOM Dispense Mapping DER Target Inlet 1 Source We Dispense Map This table shows you where the samples and detectors are on the chip Target Plate This is a graphical representation of the plate into which the samples and or detectors are pipetted 5 Click Begin Editing in the recording control pane Recording Control Playback Control BOOM C Fluidigm Real Time PCR Analysis Software User Guide 41 42 a Click the first cell from the Source Plate Then click the location in the Target Plate b Continue clicking appropriate cells from the Source Plate to the Target Plate until your custom loading map has been recorded NOTE When you click Begin Editing the dispensing pane becomes inactive 6 Click Stop Editing Fluidigm R
58. A Synthesis Kit Invitrogen PN 11754 250 SsoFast EvaGreen Supermix with Low ROX Bio Rad Laboratories PN 172 5211 e SUPERase In RNase Inhibitor Ambion PN AM2696 e 2X Assay Loading Reagent Fluidigm PN 85000736 e 20X DNA Binding Dye Sample Loading Reagent Fluidigm PN 100 3738 e DNA Suspension Buffer 10 mM Tris pH 8 0 0 1 mM EDTA TEKnova PN T0221 e Exonuclease New England BioLabs PN M0293S or M0293L e 0 5M EDTA pH 8 0 Invitrogen PN Am9260G e T4 Gene 32 Protein New England BioLabs PN M0300S or M0300L TagMan PreAmp Master Mix Applied Biosystems PN 4391128 e NP 40 Detergent Surfact Amps Solution Fisher Scientific PN PI 28324 or Thermo Scientific PN 28324 e Nuclease free Water Teknova PN W3330 Fluidigm Real Time PCR Analysis Software User Guide Preparing the Reverse Transcription RT Reaction Assembly Required Equipment e BioMark or BioMark HD System e IFC Controller MX for the 48 48 Dynamic Array IFC or IFC Controller HX for the 96 96 Dynamic Array IFC e Standard 96 well thermal cycler e 96 well plates that are compatible with the FACS instrument if FACS sorting and thermal cycler e Adhesive plate seals Applied Biosystems PN 4311971 Software Requirements Fluidigm Real Time PCR Analysis Software v 3 1 3 or higher and Fluidigm Data Collection software v 3 1 2 or higher is recommended for this protocol For earlier versions contact Technical Support Call 1
59. B 14 15 16 44 decida RO Slope y 1 95x 22 18 R 0 5618 Analyze a chip run using one of the Ct threshold 32 00 m methods below If you choose a Manual method 4 00 you can enter the desired Ct threshold s 31 30 00 Quality Threshold 0 55 29 00 Baseline Correction Linear v 28 00 Ct Threshold Method User Data Global v 27 00 o Threshold FAM MGB 0 03 26 00 Threshold VIC MGB 0 03 E 24 00 23 00 22 00 0 0010 0 1000 0 0001 0 0100 1 0000 Concentration Ready 118 Fluidigm Real Time PCR Analysis Software User Guide b Click either Pass or Fail on the secondary tool bar Fail turns the points red Pass turns the points blue The corresponding calibrator in the Calibration Table changes its call accordingly and the Call column is updated Manual Fail selected CR Analysis Tools Help 1131055065 3 Show Selected Rows Ow Style Calibrator Count Sec hie Weighted Linear 54 Ib Std6 516 Weighted Linear 7 sae siz Weighted Linear a istae sie Weighted Linear 54 Std5 513 Weighted Linear 72 1 a fan std5 st4 FYGUS Weighted Linear 72 d i FVHPR Weighted Linear 7 CO FVPGK Weighted Linear A 4 om NRC Weighted Linear CE dt 5U Weighted Linear 72 Std4 512 gt VicGUS Weighted Linear EM sd3 507 E VicHPR Weighted Linear 72 Std3 S08 VicPGK Weigh
60. Chip Run Data in the Calibration Curve View Using CCVM to Determine Concentration Levels of Unknown Samples The objective of using CCVM is to set up a chip run with at least two standard type wells where the concentration of DNA is known and to predict the concentration of the unknown type samples Four to six standard type samples are recommended The calls of experiments that include standard samples are then plotted on the graph pane CCVM also allows you to modify calls associated with calibrators This action is the same as modifying calls in the other views but also has effect of adding or removing datapoints from the regression line calibration curve calculation When first launched CCVM displays the contents and calibration curves of the data These curves are created with a default fitting method weighted linear 1 Inthe Primary View Tool Bar select a probe type such as FAM MGB 2 Onthe CCVM page click on a detector in the Detector Table pane The Detector Table displays the attributes of a detector in three columns Name of detector Style of fitting method and Count of Calibrators for this row If detectors are named the same name they are listed on one row in the table and the total of all calibrators are listed in the third column 3 Adjacent to the Detector Table is a list of the calibrators applied to that detector In the graph area below valid detectors are plotted The Calibrator Table displays six attribut
61. Dra 050 050 4 DD 8 36 E Cycle Exporting Data You can export analysis data from the Heat Map and the Results Table views The data are exported as Comma Separated Values csv files that can be opened in Microsoft Excel NOTE The data look different in the csv file depending on the view from which you exported Exporting Data from the Results Table 1 While in the Results Table click the Export icon or go to File gt Export The Export Analysis Result dialog opens Fluidigm Real Time PCR Analysis Software User Guide 101 Viewing Chip Run Data in the Data Analysis Software Export Analysis Result Save in G Desktop v Q i pe m amp My Documents My Computer My Recent my Network Places Documents My Network Save as type Table Results csv j 2 Name the csv file that is the data you are exporting 3 Navigate to the save location 4 Click Save when you are at the save location Export Analysis Result Save in O New Folder My Recent Documents File name Name the file csv k My Network Save astype Table Results csv v j Opening Exported Data csv files 1 Double click the saved csv file of interest Mame khe File cov The exported csv file below was saved from the Results Table view 102 Fluidigm Real Time PCR Analysis Software User Guide Opening Exported Data csv files 11
62. ES zr Views C e Iac Seta oD Decree BY LIEI LD det etm eth eto 0 95 SuDOd URS 088 4 SMA PESXPTI IS M 22 CO PET tr M Wow gud 404 6440400 4044 Arka frr Araia baton la anale a chp ran Bla bcc the boni Uma cr Mni males pom icai cs Changed Airis tating EOC recta ct EEE Record ot EEE Snake a chp ears ume one of Pap C1 Peri methods baia Bao chona a Maral me 7 rl AS dered C1 Peace FanchEIT1214 15 Dagas Ja T A ee ap 12 Rr put Jex3 Lien Uam Data B Fluidigm Real Time PCR Analysis Software User Guide 121 Viewing Chip Run Data in the Calibration Curve View 122 Fluidigm Real Time PCR Analysis Software User Guide qPCR Melting Curve Analysis Introduction to qPCR MCA Chip Runs eee eee Running a Chip with a qPCR MCA Protocol 0 200005 The Im Ranges ausu x doe Rd d qo eR Bh ee dex s Viewing the TM Ranges uua acaso do k coro GEO SEES eon dede dede d Editing the THPBATIOBS 4 4 4 acad ok dedo eq o 99 0x 4 3 4 4 9e ded OOH OS Working with VIBWE 44 85 wu R3 EE 43 993933 X AR RECO Results Table VIEW 0 ce RR o sa Heat Map VIG 6442 dow Rex Oe dex b Yo boa ara xe dca NI d Fick ae ee 44 4394243 452453T 123244 134 043 4 0 293222 9 54 The Mie Graph aea o Reebok oe wa VPE uy E E 3 BCH Dat us uod or de do e dE nV SERE 3d o3 X X 9 X EN Fluidigm Real Time PCR Analysis Software User Guide 123 qPCR Melting Curve Analysis Introduction to qPCR MCA C
63. ExTaq Unknown 1 00 PREP Test FBX032 Takara ExTaq Unknown 1 00 IM Test FBX032 Click the Analyze button To analyze a chip run file for the first time or After analysis parameter is changed 596 A10 Takara ExTaq Unknown 1 00 GAPDH Test FBX032 Recordiof9216 gt gt i lt E 2 Threshold Show No Call 8 Log Graph Full Range 4p Pass gFal M No cal A Clear zl Analysis Settings qPCR Analyze a chip run using one of the Ct threshold methods below If vou choose a Manual method you can enter the desired Ct threshold s Quality Threshold fo 65 Baseline Correction Linear Ct Threshold Method Auto Detectors v No Data Available No Data Available FAM MGB 4 Click Analyze IMPORTANT You must click Analyze each time you change parameters A reminder dialog appears if you do not click Analyze after each change The first time a chip is analyzed the chamber finding algorithm locates the chamber boundaries of each captured image This may take some time Continue with Setting Up a Sample Plate on page 33 Finding Corners Manually During the first analysis if the chamber finding algorithm cannot locate the four corner cells of the chip an error message appears asking if you would like to manually find the corners HiaM ark 1 J Comers in the chip weren t detected Would you like to manually find them
64. Fluidigm USER GUIDE y Layur Inlet based View Detector Inlet Position m m 00041152 TRAC 0 004112 TRAC 00041152 TRC 04013712 TFRC vogsog wu ejdures F EE Togge Threshold _ Toggle Edt ISK Toggle Log Graph C Ful Range gp Pass gral Clear Ji Ges Al FAM MGB Chamber ID Sample Name PAcDNABioChBati Detector Name LODHA Ct Call Normalized intensity Ameiticeson Normalized rtensty BIOMARK HD Fluidigm Corporation All rights reserved Limited License and Disclaimer for Fluidigm Systems with Fluidigm IFCs Except as expressly set forth herein no right to copy modify distribute make derivative works of publicly display make have made offer to sell sell use or import a Fluidigm system or any other product is conveyed or implied with the purchase of a Fluidigm system including the BioMark System EP1 System FC1 Cycler or any components thereof and Access Array IFCs Dynamic Array IFCs and Digital Array IFCs integrated fluidic circuits microfluidic chips with or without a carrier IFC controller software reagents or any other items provided hereunder This limited license permits only the use by the buyer of the particular product s in accordance with the written instructions provided therewith in the User Guide that the buyer purchases from
65. Fluidigm or its authorized representative s Except to the extent expressly approved in writing by Fluidigm or its authorized representative s the purchase of any Fluidigm product s does not by itself convey or imply the right to use such product s in combination with any other product s In particular i no right to make have made or distribute other instruments Access Array IFCs Dynamic Array IFCs and Digital Array IFCs software or reagents Is conveyed or implied by the purchase of the Fluidigm system ii no right to make have made import distribute or use the Fluidigm system is conveyed or implied by the purchase of instruments software reagents Digital Array IFCs from Fluidigm or otherwise and iii except as expressly provided in the User Guide the buyer may not use and no right is conveyed to use the Fluidigm system in combination with instruments software reagents or Access Array IFCs Dynamic Array IFCs and Digital Array IFCs unless all component parts have been purchased from Fluidigm or its authorized representative s For example purchase of a Fluidigm system and or the IFC controller conveys no right or license to patents covering the Access Array IFCs Dynamic Array IFCs and Digital Array IFCs or their manufacture such as 6 408 878 6 645 432 6 719 868 6 793 753 6 929 030 7 494 555 7 476 363 7 601 270 7 604 965 7 666 361 7 691 333 7 749 737 7 815 868 7 867 454 7 867 763 and EP Patent No 1065378 Fluidi
66. L LEE 1T 43 17 51 17 52 vii 6 IFA Ta Lx Tan 17 45 1r 4 17 47 ta Lhe 17 45 1T AH 17 64 17 54 18 7 17 32 17 32 17 26 17 37 17 33 Wa 17 23 17 33 17 4 17 31 17 38 17 35 17 35 H 7 17 33 Tx 17 41 17 28 LE Tr 17 32 iF a LECT 17 38 17 34 17 36 q 17 28 175 IT 34 17 35 17 44 17 45 17 52 1738 17 43 17 32 17 43 174 17 33 E it 1754 17 4 Fa 17 37 17 36 1738 17 38 17 46 173 17 47 1735 172 17 34 F 11 17 54 1245 UF 44 1252 17 42 UF ae a 17 45 17 41 Ws VF 41 he 17 44 Fi Fi vae Wim iM TOS 1742 Wy TOS Wal i 1 Ta 173 Lie 17 24 14 LEE TX 175 17 Va WM 17 88 T 172 17 34 7 Lhe 17 27 28 14 Tar Fal he 17 8 173 EG 17 74 Tm 173 17358 TX Tan 17 31 27 15 17 4 17 4 IT A 17 38 17 35 LEE 17 38 17 28 17 34 17 35 17 35 17 34 17 34 28 1B 17 52 17 48 17 AB 17 55 17 45 VT dal 17 44 1738 iF 41 17 32 1735 1733 17 4 Fl i 17 Ba 17 48 i755 17 51 17 55 i as 17 a8 17 55 i 3 i a 17 34 17 41 17 38 3 18 17 55 17 53 17 55 173 1736 17 44 173 17 43 7x 1733 17 44 172 17 36 Ei 19 Ta Wd iF 3 WF dz ve iF a Ws T3 7 1 War 2 V Tx EJ m 17 SF Waa Va 7 44 Vs ia 17 27 Waa LE 17 0 TX Lie Vs EJ z Tar 17 25 iF BF TF be Ba hard 17 4 T3 LE Fs 17 82 IT Lk rd 173 ET m 18 76 18 02 1B ee 18 17 B4 1T B 1781 17 85 1756 17 88 18 4 N55 3m 35 4 TA 17 38 172 17 28 1732 28 173 17 28 iF af 173 1721 i 2 17 24 36 7 17 45 17 34 MT 3 LAE 17 15 i 26 17 31 17 3 LEE 17 35 1T 45 1232 17 35 37 Fl 15 52 16 34 i5 ax i53 1624 th 3s 16 27 1827 th 2 1E 15 16 19 1 amp 2 ib 2 EJ 5 1525 E
67. Pre Mix and Samples 1 Prepare a Sample Pre Mix solution containing the Master Mix and 20X GE Sample Loading Reagent sufficient for the number and type of chips to be run The following table provides the component amounts for one 1 48 48 or one 1 96 96 chip Volume per Inlet with Overage pL Volume per Inlet pL Sample Pre Mix Sample Pre Mix for 48 48 pL for 96 96 pL Component A 2X Master Mix B 20X GE Sample Loading Reagent Diluted RT STA Sample Total Volume Quanta PerfeCTa qPCR Fast Mix low ROX Quanta BioSciences PN 95078 012 or VWR PN 1014190 220 Table 4 Sample Pre Mix and Samples These volumes include some overage to account for pipetting error 1 Ina DNA free hood combine the two Sample Pre Mix components A and B from the table above in a 1 5 mL sterile tube enough volume to fill an entire chip Aliquot 3 3 uL of the Sample Pre Mix for each sample to be analyzed in an empty 96 well PCR plate Fluidigm Real Time PCR Analysis Software User Guide 165 2 Remove the Sample Pre Mix aliquots from the DNA free hood and add 2 7 uL of the diluted RT STA sample to each to make a total volume of 6 uL then seal vortex for 10 seconds and centrifuge at 1500 RPM for 1 minute Priming and Loading the Dynamic Array IFC For instructions on loading the 48 48 Dynamic Array IFC see Fluidigm 48 48 Real Time PCR Workflow Quick Reference PN 68000089 For instructions on loading the 96 9
68. R Analysis Software User Guide 65 Viewing Chip Run Data in the Data Analysis Software Unsorting Columns 1 Right click a sorted column header 2 Click Clear Sorting Chip Explorer 5 gj Chip Run Gene Expression A Analysis Views B m Sample Setup is Sample Mapping View B Detector Setup Column Chooser Analysis Views HB Bj Results Table d Show Selected Row Drag a column header here to group by that columr Experiment Information Chamber Sample Sort Ascending P Sort Descendin R48 C02 4l Clear Sorting R48 CO3 R48 CO4 Group By This Column R48 C05 Group By Box R48 CO6 4A Column Chooser R48 CO 3 Best Fit R48 C 8 R48 CO9 R48 C10 Best Fit all columns NAO oii cni Depending on how you set up your sample plate and detector plate you can have 204 columns in the Results Table all of which are not viewable at once To temporarily remove columns not of immediate interest follow the procedure below 1 Right click a header 2 Click Column Chooser The Customization dialog opens Chip Explorer 5 gj Chip Run Gene Expression A Analysis Views m Sample Setup is Sample Mapping View B Detector Setup Analysis Views E EE Results Table Show Selected Rows Drag a column he group by that column in Experiment Information Chamber Sample ID Mame x ROI C32 548 2l Sort Ascending ROI C33 548 Z Sort Descending RO1 C34 548 RO1 C35 548 Group By Th
69. S 152 1 amp 2 1626 16 8 156 152 a 1528 EN 152 ES E a 1535 1641 X 1525 153 GS 163 EN 63 6S 163 be OGR E Fai 7504 T 27 6 27 16 32 15 34 Lm T5 24 16 25 15 18 16 27 16 32 12 1633 41 F 16 35 16 28 LR n TE 25 16 34 vb 18 15 38 1x TE 26 15 2H BA o 1828 42 n 16 16 82 15 45 15 25 1635 ib LT 16 35 15 3 16 1 EH 45 5 1528 43 3l 15 41 15 44 t5 47 15 38 16 38 15x 15 37 1633 15 35 15 32 l amp s 35633 1E 27 44 X 15 51 15 38 i5 bl 16 38 1638 th 3k 15 34 1E 4 ibas 1E 3 16 34 Lu 15 25 45 3 15 di 16 57 i5 A x nj 16 35 th 31 15 3 16 38 ib 2 tb 3 1a 3t 15 31 1E 33 ca 34 1S 1643 1653 OES ES 163 EN 62 16S 6J IES 75 5 4T x 1538 1632 tH 1633 153 X 15629 1632 163 1637 UES 36507 N ait x 16 54 15 35 vb s LR 15 35 ue 181 15 XT vb Tb 1B X3 T5 1631 48 x 16 4 15 35 DES 151 16 32 rb s TE 42 1E XT vb a 16 16 16 32 EJ 38 15 33 16 38 16 36 1E 35 1E 37 EE 15 38 1638 ED Ak EF 15 18 16 24 51 E i53 15 3 IE i15 xs 16 45 tB ar th 3 1638 ib3r Ha 15 33 15 31 15 23 52 An 15 3 15 3 IE 16 33 16 38 th ddl 16 65 16 43 that iE 33 1629 1b 1E 35 EJ at i537 1B 3 SF ST i54 i543 1546 13545 1638 ET a2 054 1543 1543 1585 RS 163 l amp 4 163 1643 164 1544 sh 43 15 23 16 41 v5 adt 16 44 18 84 vb as 1 166 fun 16 45 16 8 16 38 16 47 EJ 44 16 163 1 amp 4 T5 24 16 54 tb os TE 4r 168 BS 16 16 64 16 T 61 87 45 15 34
70. T T1 T1 T TL TF EF TL Pat CEE ieee 2 reire elta Delta C Heat Map Data with Inlet Based View Reference cells is Analysis Views HD Bil Heat Map view dal Show Selected Cell m 9 ram mas Delta Delt v Inlet based view FAM MGB Ct A idi pee FAM MGB Delta Ct Samp Detector Inlet Position WM 2 R B s a 8 SiAn Man Deka ct Reaal EERE EEEBEEREBREEEEEEEGEEBEE EEE A A FAM MGB Delta Delta C Heat Map Data with a Chip Based View Reference cells B Analysis Views p NH Map view ee Sho T ted id LER Dj w z FAM MGB Delta Delt m Chip based View m Inlet based view bro ctor Chip Position R 8 8 8 9 8 8 aj s Ex s ni SY OS 81 e E En SY b e in ls 43 redere is m E w 48 retre 45 refer E Se Chip based View BERBER RPE PER SPER E REE a T aig E Er ER TE s SD E FR BR T TUER MA retre 43 retre H2 rere 41 rere MO retre BS restore 38 retre B7 refere 35 rere 35 rere H retre 33 rere R2 retre L T gs Congratulations you have successfully viewed your analyzed chip run data Fluidigm Real Time PCR Analysis Software User Guide 111 Viewing Chip Run Data in the Data Analysis Software 112 Fluidigm Real Time PCR Analysis Software User Guide Viewing Chip Run Data in the Calibration Curve View ie Ls rm A Poe ee on os 39 3409 ERE eee ERA EE RRR eRe ee Using CCVM to Determine Concentra
71. Than One Cell Isolate data for a single cell or for multiple cells in any analysis view Results Table Image View or Heat Map using the following methods 98 Fluidigm Real Time PCR Analysis Software User Guide Using the Graph View In the Analysis Views Window The Results Table Procedure Press and hold the keyboard SHIFT key and click the 2 outer cells for a continuous range of cells The data for the range of cells display in the Graph Views Or Press and hold the keyboard CTRL key while clicking individual cells Example ih ae eS a a awe zum mom z CN L Le L tL e n N GO U L T L imm mm Am 753 hapi WE umm e um n 329 jj 3S uoo pes i Image View Press and hold the keyboard OQ CTRL key while clicking fr E Image View Shi individual cells of interest a i da You cannot select a E LOU m E contiguous range in this view Heat Map Click a cell and then hold and drag to highlight a range of cells Or Press and hold the keyboard CTRL key while clicking individual cells Or Click on the Column or Row heading and select all chambers in the column or row m B Fa amp 9 ram mes ct Peres m RDA CA00000 BOBO EERE ES d HEB NE 05 505 O68 S06 yu ajdwes Fluidigm Real Time PCR Analysis Software User Guide 99 Viewing Chip Run Data in the Data Analysis Software Cross Highlighting and Sele
72. This document provides a fast cycling protocol that can be used on either the BioMark HD with fast ramp rates 5 59C s or the BioMark with the normal ramp rate 29C s This protocol can be used with the 48 48 Dynamic Array integrated fluidic circuit IFC or the 96 96 Dynamic Array IFC The use of the fast ramp rate on the BioMark HD System requires the use of a PCR master mix that has been optimized for fast cycling The fast master mix recommended for use in this protocol includes both EvaGreen and ROX in the master mix which makes it convenient to use This master mix also works well on the BioMark System with the normal ramp of 29C s The total cycling time on the BioMark System will be longer than the cycling time on the BioMark HD System but still faster than standard protocols Primers need to be designed to reduce the potential for primer dimer formation and to be highly specific for the target of interest For the development of this protocol we used DELTAgene Assays a set of assays designed by the Assay Design Group at Fluidigm which avoid SNPs and are highly specific for the gene of interest We recommend the use of specific target amplification STA to increase the number of copies of target DNA Prior to qPCR reactions the STA reaction is treated with Exonuclease to eliminate the carryover of unincorporated primers References 1 SsoFast EvaGreen Supermix With Low ROX product literature http www bio rad co
73. Viewing Chip Run Data in the Data Analysis Software Analysis Views 230 255 H 425 230 285 H425 m 555 M 695 H5 H 345 E 1075 EP 1465 E 1535 H 25 EH 18 55 EH 1395 H25 B 3570 uonisoq18ju ejdue s Preferences Click the Preferences button Show Grid Show Cell Text Show Cel Text Preferences Ira Changing Grid and Selected Cell Color Preferences To change heat map grid lines 1 Click Preferences 2 Click the Grid line color rectangle The color palette opens 86 Fluidigm Real Time PCR Analysis Software User Guide Using the Heat Map Preferences Preferences Grid Preferences Show Grid Grid line color Grid Preferences Show Grid Grid line color Selection frame color Selection frame color Custom colors IE EOS S SS SN BEBE Hf Define Custom Colors gt gt Click on a color Click OK Click the Show Grid box Click OK The new color grid lines display in the heat map a U A W Analysis Views HD Bil Heat Map View show Selected Cell E mh m mel A amp fal rit Eva Green at 20 C Ct Layout Inlet based View z Detector Inlet Position RJE R z tReet H ts td d ta al BSEEL Reese cR EE Ee DE PRRRR RRRERERERRRE 51 351 07 507 03 50 E L amv om fa o9 07 307 oe sce sss T5515 EEE Dea 13 513 4 14 15 515 Teste 17 517 Teste T5 515 25 975
74. a EH EHE HERE EH E REESE EE EE REP E EE EE E EE EE Pr ERE Ek a bb EEEE EEEE EEEE EEEE EE EHE EHE EEEL HEEE ENEE EHE EHE ELE EHE ERE EEHFLBEEHTECTEETETEH PEHPEEH T EETTPETET EH TETTE EHE PET EETTT EH PEDE E EET EL TTELTEL ER T LET EET PED UON frugum Anian bair To analyse a the run Me har tha firit Gria or After analetit parameber ap changed Arabis Setri FR MEA Janus a chip nun wig one of fee Ct Deed bold Pahadi baii V pou chocdbe Mania method you can nber ia dened Ci thresholds Chasity Thrashed nis Emmm Cone fers Leto D TWweshokiMeHhed Auta ikbal Ho Dois Loan Pio uis iyasi m Eva rsen at 20 C HEA NOTE A black square indicates no C value or a value outside of the spectrum range as shown in the example below Also negative controls that do not show amplification appear as black squares Chamber ID 12 417 Sample Name 512 Detector Name amp 17 Ct 999 00 Call fail Tm 333 00 NOTE An X signifies an amplification curve marked as fail 3 Optional Click the double arrow EI to expand the image 76 Fluidigm Real Time PCR Analysis Software User Guide Using the Heat Map 4 Optional Hold your cursor over a cell of interest and an information dialog box opens click the cell and the information appears on the task bar Hold the cursor over a cell to open the information dialog box 02 502 X 03 803 Chamber ID S01 A01 Sample Name S01 04 504 D
75. able after a chip run bml file has been opened and analyzed in the software Fluidigm Real Time PCR Analysis Software User Guide 27 Install Test Report 1 Select Report gt Install Test Report A warning about properly setting up the chip appears Click OK if the setup is correct The report is generated Preview 4 ILJ X fle yew Back round S O 1a amp De ey amp 10 V amp IP amp gal a x Fluidigm H GEN amp T 1 ANALY S 1S i Real Time PCR Analysis Test Report Barcode 1361440131 ichip Type 96 96 136x File Location C Documents and SettingsWorenda jordan DesktopVl 361 4401 31 ChipRun bml Protocol PCR Passive Reference ROX Probe Type s FAM MGB Experiment Time Start Duration 0543 2010 16 52 01 0 28 23 System ID BIOMARKLFFO1 l Application Version Build 3 0 4 2010061 4 0553 l Comment Quality Threshold 0 65 Baseline Correction Method ART EI aed ril y aar Dutlook i Fluidigm Real Time PCR A J aec wc Go to File Export Document to select a file format PDF is the default Select a folder location to save the file Change the Name and or file type if needed Click Save gi A W N Tools The Tools menu has the following options Tools Dispense Map Editor Options See Using the Dispense Map Editor on page 40 28 Fluidigm Real Time PCR Analysis Software User Guide Creating a New Chip Run Cre
76. ai mew Chant Cal E Cercs Maecen urn T TT fest ILF 0n T 14 an wf 1 00 Test fest 23 1 isa w TE Ls w 1 50 Test Peat 3 w 38 42 ud dl 1 08 teat feet EEE i jd 6 35 wt Len Tid Teil 2127 tm wt TET 0 71 wt LE Test Teri 11 4 54 wf w M DET wr Lon Tet Test 24 57 u Ca 144 OAT al LE Test Teri 23 73 Pr w Ac o m w 100 Test Teri Hy n w TT au a L Test Teri LH wt La uas yf Lo Test Teri LH Uu mi TET on x 1 00 Teak Teri 4 D wf 25 4 Da Lt d L Te Teri x LT 144 0 81 nd aen p L lian A Tw pun what s x To vob cd ec fil rin nd rn Lon Test fee nna Ls wf pE a Mer acp paracutbe i arge t ferit Tei 19 ta wf 1 fu m Lon Tit few 5 41 t w 23 t a Loe teat Pent 23 oe w 1 1 LE w Anian Taeg Li mi fest 23 38 el w 31 3 tual ww Avalos a chip san unire ores cd Ba Ct Bopha w E pietre amd i E T murs m d yu T sung Eder coder ee ied C1 Pheerbedi OOD Record Vel INH nc 2 Cni Toi DT here EA Tog Leg Gragh wf Fase Mrs e Pi EE WC ee Chamber If 48 A15 Sample Mame del Detector Name Ch 33 03 Gk a Bauer cocta Lr r Raw Daa Da ANT i Dinha Meticct Hado ital E Sango 06 oH Tee 06 f 5686 LAT nee de dene o0 DE cese poe 3 Bi 1200800 n 22x60 oo gt wun S t E ote deca b S008 0n ot 300 06 Lon lip ee i amp 1 odi 3s X ny s ou i 8 70 cT n xw non y au amp Cacie Dros Probe specific graph tabs NOTE You can use the Call Redo or Undo buttons GLO to revert back to the original call state
77. and 48 TaqMan assays and allow them to be systematically combined into 2 304 reactions Fluidigm Real Time PCR Analysis Software User Guide 17 BioMark System 96 96 Dynamic Array IFC for Real Time Quantitative PCR The 96 96 Dynamic Array IFC is a matrix of channels chambers and integrated valves finely patterned into layers of silicone Valves within the array partition 96 samples and 96 TaqMane assays and allow them to be systematically combined into 9 216 reactions The following table illustrates the advantages of Dynamic Array IFCs compared to microwell plates using TaqMan assays Dynamic Array IFC Features Dynamic Array IFC Benefits Lower running costs Saves on reagents and pipette tips and on the Nanoliter reaction volumes upkeep of liquid handling robots Higher throughput The 48 48 Dynamic Array IFC provides 2 304 data points per run The 96 96 Dynamic Array IFC nlgn density reaction Cnamvels provides 9 216 data points per run More Informative Generates multiple readouts per sample without the spectral overlap and cross amplicon influence An N x M rows by columns architecture of multiplexed PCRs coupled with nanoliter reaction volumes Highly Flexible Facilitates the input of any set of samples and any set of primers probes detectors delivering the Input frame with microwells together throughput of a fixed array with an N x Marchitecture After pipettin
78. are User Guide 149 Priming the Chip and Loading Assay and Samples CAUTION Due to different accumulator volumes use the appropriate control syringe for your chip type 300 pL for the 48 48 Dynamic Array IFC or 150 pL for the 96 96 Dynamic Array IFC 1 Inject control line fluid into each accumulator on the chip see Figure 1 for the 48 48 Dynamic Array IFC or Figure 2 for the 96 96 Dynamic Array IFC 2 Remove and discard the blue protective film from the bottom of the chip 3 Place the chip into the IFC Controller MX for the 48 48 Dynamic Array IFC or the IFC Controller HX for the 96 96 Dynamic Array IFC then run the Prime 113x script for the 48 48 Dynamic Array IFC or the Prime 136x script for the 96 96 Dynamic Array IFC 4 When the Prime script has finished press Eject to remove the primed chip from the IFC Controller CAUTION While pipetting do not go past the first stop on the pipette Doing SO may introduce air bubbles into the inlets 5 Pipette 5 uL of each assay and 5 uL of each sample into their respective inlets on the chip 6 Return the chip to the IFC Controller 7 Usingthe IFC Controller software run the Load Mix 113x script for the 48 48 Dynamic Array IFC or Load Mix 136x script for the 96 96 Dynamic Array IFC to load the samples and assays into the chip 8 When the Load Mix script has finished remove the loaded chip from the IFC Controller 9 Remove any dust particles or debris from
79. at the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or the Licensing Department Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 2 Contact Fluidigm technical support to discuss your non fluorescent quencher requirements Fluidigm Real Time PCR Analysis Software User Guide 21 BioMark System Sample Requirements DNA Quality Your cDNA should have an 260 280 Ratio between 1 5 and 1 8 Prior to use ona chip monitor the integrity of your cDNA on a system such as the Agilent 2100 bioanalyzer CDNA Input The exact amount of cDNA to be used for each experiment depends on the relative abundance of the target gene Unless you have concentrations in excess of 1 000 copies of your target template per ul of sample we recommend that you increase the your target concentration by using specific target amplification as described in Fluidigm Specific Target Amplification Quick Reference Card PN 68000133 cDNA Storage Avoid multiple freeze thaw cycles by storing cDNA at 49C For longer storage aliquots may be stored at 209C Reagent Storage Consult manufacturers product inserts for storing specific reagents 22 Fluidigm Real Time PCR Analysis Software User Guide Using Real Time PCR Analysis Software LOC The Se a adde aes aede a OA OER ATER OHS PEO enl 24 Menus and IE auo se Se ad ee XX X 30 4 X39 9 ee eee hE 24 Creating a New Chip RUN 1
80. ating a New Chip Run 1 Click creating a new chip run under Chip Run Summary or click Create a New Chip Run under Task IFluidigm Real Time PCR Analysis E File Edit view Report Tools Help O A5 w 9 Chip Explorer Chip Run Summary 3 Chip Run Summary E El Fluidigm uidigm T5 a GENETIC ANALYSIS Welcome to Fluidigm Real Time PCR Analysis Software Start by opening an existing chip run or creating a new chip run Quick Tasks QJ Open a Chip Run Create a New Chip Run Recent Chip Runs C 5 1361440131 ChipRun bml C 5 41361355177 ChipRun bml C 5 41351402100 SChipRun bml C 5 41131055055SChipRun bml C 5 4888822001 1 GE SChipRun bml C 5 41130212155SChipRun bml C 41131054061 GE perf ChipRun bml i U a QU QU dad Ready The Chip Run Setup Wizard opens 2 Follow the steps to complete the setup 3 Complete the wizard and go to Setting Up a Sample Plate on page 33 Opening an Existing Chip Run 1 Toreanalysize a previous chip run click open an existing chip run under Chip Run Summary or click Open a Chip Run under Task or click File Open Fluidigm Real Time PCR Analysis Software User Guide 29 Fluidigm Real Time PCR Analysis P nf File Edit view Report Tools Help Daid Cnr Our Di 2 Chip Explorer ve di Chip Run Summary Chip Run Summary Fluidigm GENETIC ANALY SIS Welcome to Fluidigm Real Time PCR Analysis Software Start byf opening an ex
81. ave Pate Puta Pak Pate p Ed 15 Fazi Paj Pait Pek Pata Pukk Pakk Poig Pakko Park Piia Part PP CI 17 Pa Page Pam Past Pug Pam Pam Pars Pap Pom Po Pam Pog P Ea 16 Pass Pass Pass Pass Pass Fass Pass Pass Fass Pass Pass Pass Pass Po tal 18 Pass Pass Pasa Pass Pass Fass Pass Pass Fass Pass Pass Pass Pass P I am Pass Puss Pass Fass Pass Pass Pass Pass Pass Pass Pass Pass Pais P i Fist Pata F ti Pad Pata Patt Pade Pais Patt Pate Pata Patt Pate P B5 2 Fui Pata Pati Pati Pats Patt Pat Pais Pipi Fa Fail Fui Fad Fi BT A Pai Pana Patt Past Pans Patt Page Pats Poet Pate Puta Pace Pate Py ca 34 Pap Pas Paus Pas amp Pa Paus Pap Pug Pag Pame Poa Pas Pags Rh E4 25 Fass Pass Pass Pass Pass Fass Pass Pass Fass Pass Pass Pass Pass P Ei zh Pass Pass Pass Pass Pass Fass Pass Pass Fass Pass Pass Pass Pass P El F Past Pass Pass Pose Pass Pass Pase Pass Pass Pade Pasa Page Pate F t 104 Fluidigm Real Time PCR Analysis Software User Guide Calculating Delta Ct Sample Values Calculating Delta C Sample Values To calculate the delta A C samples 1 Click Sample Setup 9 BioMark Dynamic Array Analysis Analysis Views Sample Setup c lH Detector Setup EZ Detector Mapping View Type Type Name Name 3 Click Editor 4 Click Reference Sample Editor Type 4 Blank Sample Name e MAE Relative Conc NTC Unknown Reference Reference 5 Enter a sample na
82. ave a plate for reuse Import to open an existing plate Plate Settings Source 96 Wellplate Name Barcode Mapping 96 96 S ample SBS96 m Sample Contents Passive ROX F Reference L Contents le ama EL i z Ready Your sample plate setup is complete Next go to Using the Sample Mapping Viewer Using the Sample Mapping Viewer Use the Sample Mapping Viewer to view or record the loading pattern after setting up the sample plate 1 Click Sample Mapping View The dispensing map opens 2 Clicka cell in the Source Plate to see where it loads on the Target Plate File Edit View Report Tools Help aN Ll ry S d x E x J hip Exploret j Sample Mapping View a Chip Run Summary 8888220011 C3 Analysis Views m Sample Setup Sample Mapping View BB Detector Setup Source Plate SBS96 Dispensing Map 96 96 Sample SBS96 Dispensing scheme from a 58596 plate to 96 96 S ample Inlets Task Setup Click one of the following New to create a sample plate Export to save a plate for reuse Import to open an existing plate Plate Settings Source 96 Wellplate Name Barcode Mapping 96 96 Sample SBS96 5 4 Sample Contents Passive Bn Reference Man ES Contents S Name Included mi r1 M L Ready Fluidigm Real Time PCR Analysis Software User
83. ble 7 Assay Mix solution 2 Vortex the Assay Mix for a minimum of 20 seconds and centrifuge for at least 30 seconds to spin down all components IMPORTANT Vortex thoroughly and centrifuge all sample and assay solutions before pipetting into the chip inlets Failure to do so may result in a decrease in data quality NOTE The final concentration of each primer is 5 uM in the inlet and 500 nM in the final reaction Priming and Loading the Dynamic Array IFC CAUTION Due to different accumulator volumes use the appropriate control syringe for your chip type 300 uL for the 48 48 Dynamic Array IFC or 150 uL for the 96 96 Dynamic Array IFC 1 Inject control line fluid into each accumulator on the chip 2 Remove and discard the blue protective film from the bottom of the chip 3 Placethe chip into the IFC controller MX for the 48 48 Dynamic Array IFC or the IFC Controller HX for the 96 96 Dynamic Array IFC then run the Prime 113x script for the 48 48 Dynamic Array IFC or the Prime 136x script for the 96 96 Dynamic Array IFC Fluidigm Real Time PCR Analysis Software User Guide 135 4 When the script has finished press Eject to remove the primed chip from the IFC Controller CAUTION While pipetting do not go past the first stop on the pipette Doing so may introduce air bubbles into the inlets 5 Pipette 5 uL of each assay and 5 uL of each sample into their respective inlets on the chip 6 Return the chip to the IFC
84. cal reference map Auto contrast adjustment Manual adjust contrast slider Dye selector Cycle image drop down menu Toggles overlay On and Off Fit image by auto width or height Zoom You can increase or decrease the image view size in several ways e Multi clicking the magnifying glass buttons and e Click the 100 button Clicking the Fit button to fit image to width Clicking inside Image View and then rolling the mouse scroll wheel up backward larger down forward smaller Fluidigm Real Time PCR Analysis Software User Guide 71 Viewing Chip Run Data in the Data Analysis Software Click and hold anywhere on the image When the hand icon appears drag the image until it is centered on the cell of interest Location Reference Map Use the location map to reference your cell of interest within the entire framework of the chip 1 Click the Location Reference map icon to open the map 2 Click and drag the blue rectangle to a location of interest In the example below the blue rectangle within the Location Reference map is dragged to the green cells which enlarges the green cells in the Image Viewer Drag blue rectangle to area of interest Dragging the blue rectangle to an area of interest enlarges that area in the Image Viewer as shown here 72 Fluidigm Real Time PCR Analysis Software User Guide Using the Image View Adjusting the Size of the Location Reference Map The size of the imag
85. ccur without the PCR working QM NTC No Template Control negative control everything included except the sample to show that a positive result cannot be obtained when the sample is left out gt Unknown An experimental sample 2 Reference A sample against which the unknown samples are compared or normalized g Standard A sample against which unknown samples are compared in a standard curve analysis 1 Inthe Chip Explorer window select Sample Setup Fluidigm Real Time PCR Analysis Software User Guide 33 Fluidigm Real Time PCR Analysis File Edit View Report Tools Help ODAH G a E e Chip Explarer El F Chip Run Summary 1361440131 L Analysis Views EZ Sample Setup Detector Setup Task nx Setup Click ane of the Follewing Hew to create a sample plate Export to save a plate for reuse Impart ta open an existing plate Plate Settings 5nurce S amp Wellae M ame PO Barcode PO Mapping Sample Contents Passive Reference Contents Container type SBS Plate v Container format S5B536 v 1 Choose the appropriate Container type and Container format Container type SBS Plate the plates where samples and detectors are stored before being pipetted into chip Sample Inlets location where samples enter the chip Container format e SBS96 a 96 well plate 2 Click OK 3 Select
86. cting When you select multiple chambers they are displayed as a line in the lower graphs Detector Inlet Pasition SE T elelelelale siele alz zla z mmm mm EESIEESEBBE zestril SERE EB E Yi ch 3B L 07 507 E ces fF eer Al FAM M B Chambar lD Sample Mama Detector Nama AID Ct Call wv To Cross Highlight hover the mouse over a graph line and its chamber will be highlighted in the other graphs diet Si Detector Inlet Position n3 ow 01 S01 b S06 07 507 09 S09 10 510 11 911 12 912 uogisoq19ju j dures 5 S g o e co Em 2 R 14 514 15 15 16 18 17 817 lt Fa E Threshold Show No Call Log Graph Full Range gp Pass ig Fail No Call Clear A FAM MGB Chamber ID Sample Name Detector Name A10 Ct Call Y Normalized Intensity 0 40 B 0 70 i 100 Fluidigm Real Time PCR Analysis Software User Guide Exporting Data Double clicking on the graph line selects it in the primary view and the graph view 1 Detector Inlet Position QE EIE SEES ERES ETESES SES ERES EE PRESE EBEEEBEBEEEEEZEEEZZERERESESESE uu gl Teeshoid F Show No Call Mil tog Graph D Ful Range e Pars ile teca D clear EY Clew A ip FAM MGS Chamber ID SD4 A1D SsmpeName SIH Detector Name A10 Ct 16 80 Cal Hormakzed irdenaiy Amg ficabon
87. ction methods contact Fluidigm Technical Support Always compare the T of the intended products to a positive control sample Click Analyze NOTE For more information about melting curve analysis see the Fluidigm Real Time PCR Analysis Software v 3 0 User Guide PN 68000088 Fluidigm Real Time PCR Analysis Software User Guide 139 140 Fluidigm Real Time PCR Analysis Software User Guide Two Step Single Cell Gene Expression Using EvaGreen Supermix on the BioMark or BioMark HD System dosis A4 DD MCN ee a he SH OER EEE AR OREO RE RS Preparing the Reverse Transcription RT Reaction Assembly Fluidigm Real Time PCR Analysis Software User Guide a Os wv te ia a E Oe Re Ee ee Preparing STA Reaction MIX uua x ae ke de 44 EE EEREG CES OE a EDGES Preparing LOX STA Primer MIX 2 ee Preparing the Sample Pre Mix and Samples 0 0 0 cee eee ees Preparing the 5 uM 10X Assay MIX 1 ce RR es Priming the Chip and Loading Assay and Samples 00 00000 Using the Data Collection Software enn Using the Real Time PCR Analysis Parameters llle 141 Introduction This protocol includes a separate reverse transcription step and a specific target amplification STA step hence its two step title The following protocol enables the use of a DNA binding dye for quantitative PCR gene expression DNA binding dyes offer flexibility at a very low upfront cost relative to probe based assays and
88. cule rather than fluorescing resulting in a non fluorescent substrate Dual labeled probes are designed to hybridize to a complementary region of the cDNA The probe is flanked by an upstream and downstream primer pair that generates a PCR product During PCR when the polymerase extends the PCR product from the upstream primer the 5 exonuclease activity of the polymerase cleaves the probe This separates the fluorescent quencher and reporter dyes and Fluorescence Resonance Energy Transfer FRET no longer occurs The increase in fluorescence intensity is proportional to the number of probe molecules that are cleaved Forward Primer Probe 5 re nal 3 3 5 P a 5 Reverse Legend Primer FAM Quencher m Reporter b MALI Target Template AmpliTaq Gold DNA Polymerase Q Non Target Template Fluidigm Real Time PCR Analysis Software User Guide 15 BioMark System BioMark HD System for Genetic Analysis The BioMark HD System includes the optical thermal cycling and software components necessary to perform real time quantitative PCR qPCR analysis on Dynamic Array IFCs The BioMark HD System provides orders of magnitude higher throughput for real time qPCR compared to conventional platforms due to its Dynamic Array IFCs nanofluidic chips that contain fluidic networks that automatically combine sets of samples with sets of assays This innovative solution for real time qPCR
89. d Reverse primer for each assay so that the concentration of each ms Q NOTE If you obtain primers from another source combine the Forward primer is 100 uM Proceed to step 1 below 1 Prepare 500 nM primer mixture 10X for Specific Target Amplification STA e Pool together 1 uL aliquots of all of the 100 uM primer sets to be included in the STA reaction up to 100 assays e Add DNA Suspension Buffer to make the final volume 200 UL e The volumes used in this step can be scaled up as needed 2 Prepare Pre Mix and Samples for STA Fluidigm Real Time PCR Analysis Software User Guide 131 e n a DNA free hood prepare a Pre Mix for the STA reaction as indicated Component Volume Volume for 48 Volume for 96 Reaction pL Reactions 10 Reactions 10 pL Overage pL 2X TaqMan PreAmp Master Mix Applied BioSystems 500 nM 10X pooled primer mixture Water cDNA 1 25 Total Volume 5 0 Table 1 STA Reaction solution e Aliquot 3 75 uL of STA Pre Mix for each sample e Remove from the DNA free hood and add 1 25 uL of cDNA to each making a final volume of 5 uL e Vortex to mix the reactions and centrifuge for 10 seconds 3 Thermal cycle the STA reactions e Place the STA reactions from Step 2 in a thermal cycler and cycle as indicated Condition Hold 10 14 Cycles Hold me ewe m e e Time 10 minutes 15 seconds 4 minutes Table 2 Thermal cycle conditions 4 Clean up the STA amplificati
90. d Selecting 00 eee ee ee ee ee ees 100 asen pt e P ee eee ee ee ee a ee er ee ee ee 101 Exporting Data from the Results Table 0 000 eae 101 Opening Exported Data csvfiles 0 ee 102 Calculating Delta Ct Detector Values 0 0 ce ee ee 109 Delta Delta Ct Values 4 4 ugue HES ee Oe OS X de x 404 Vea 110 Viewing Delta Ct Data in the Heat Map 00 eee eee 110 Fluidigm Real Time PCR Analysis Software User Guide 53 Viewing Chip Run Data in the Data Analysis Software Working with Analysis Settings You can customize these analysis settings e Quality Threshold Baseline Correction e C Threshold Method To change the settings 1 Launch the Real Time PCR Analysis software 2 Click Analysis Views Analysis settings are located under Task d F baidiga Rost Time PCR Analysis fie Dd Vew Repat Tode Heb Chip Pun Summy N Lau Rl ut arn w Sante Sup Jatt aig Gligeem Uripa 100 10 116 mou GT petes Lines t PREP Pao GYyrpeess Uripa L00 VIN FEL 50 10 GU pes Lr oomn Ln aero ANR 306 A11 Glypee s Urine 109 reer ice FEWR 506A 12 GT quee Urine LOD PPLA nn 39415 Gres Urina 100 reer FEON EE a oue urn 100 QN mna fando amp ay tick te Aenda teten 395 A1 gt Des Uden I 0 I4 1 Pron To anayo chap vun Me Vor he feti tree c 50A M6 GTqpeeec Urine 10 APPO ro Ale eran potant et i charged 905 A17 Gees L on 1400 cool FEDR aan GIyever Lino 14
91. e Melting 81 Temperature Editing the Tm Ranges Toggle the Threshold Edit button to display the MCA tab and directly change the T ranges for each detector Fluidigm Real Time PCR Analysis Software User Guide 125 qPCR Melting Curve Analysis You can also click and drag the T ranges Melting AR dT Temperature Working with Views There are three views e Results Table Heat Map e Graph Results Table View A T4 section appears for each probe type The first column is the value for the T4 peak detected in range The second column is the value for the out of range Tm peak A value of 999 means no peak was detected Tm In Range Out Range Peak Ratio Peak Cal 7 71 39 999 00 1 00 v 74 10 999 00 1 00 v 74 73 999 00 1 00 d 82 03 999 00 1 00 ad 82 20 999 00 1 00 v Heat Map View For MCA runs there are two additional data views Inside T and Outside T Also the spectrum is adjusted for T values EvaGreenat20 C Ck Eva Eva Green at 20 C Inside Tm Eva Green at 20 C Outside Tm Graph View In this view there are two layouts Combined and Tabbed 126 Fluidigm Real Time PCR Analysis Software User Guide the Combined view shows the visible Amplification C and Melting Tm graphs E Ld Combined a Log Graph ge i 8B Fail io No Call i 5 Eva Green at 20C Chamber ID Sample Name Detector Name NC_004741 50194 ct Call Tm 999 00
92. e B Reference reference 00001 reference 00001 reference 00001 4 a d wd Unknown Unknown Unknown Name Name rConc rConc Ref Ref Unknown Type Unknown Type Unknown Name Name rConc rConc Ref Ref Unknown Type Unknown Type Unknown Name Name rConc rConc Ref Ref Unknown ype Unknown Type Unknown Name Name rConc Ref Unknown Unknown Type Unknown Name Name rConc rConc Ref Unknown ype Unknown Type Unknown Name Name rConc Ref Unknown Unknown Type Unknown Name Name rConc rConc Ref Ref 8 Click Editor 9 Enter a sample name 10 Select 11 Select Unknown the reference you created Sample Editor x Quim vj reference to reference 00001 Tew RE Fluidigm Real Time PCR Analysis Software User Guide reference OOO NS 00001 M Bj 0 VL Type Name rConc Ref Type Name rConc Ref Type Mame rConc Ref Type Name rConc Ref Name rConc Ref Type Name rConc Ref Type Name rConc Ref 1 J mm own Unknown Unknown Unknown Unknown Unknown Unknown Unknown Type Name rConc Ref Type Name rConc Ref Type Mame rConc Ref Type Name rConc Ref Type Name rConc Ref Type Name rConc Ref Type Name rConc Ref 107 Viewing Chip Run Data
93. e in the Image Viewer determines the size of the blue rectangle in the Location Reference Map In the example below left the image is has not been zoomed so the blue rectangle on the map is large In the example below right the image has been enlarged by clicking in the Image View and then rolling the scroll wheel on the mouse TL Le tage re F Sem Sette 000000 Box Here the blue rectangle is large and cannot be dragged with much accuracy Here the image has been enlarged so that the blue rectangle is smaller and easily dragged to an area of interest E oS NOTE Selected cells in the Image View are also displayed in the Graph View as shown below Fluidigm Real Time PCR Analysis Software User Guide 73 Viewing Chip Run Data in the Data Analysis Software E lt mage View ded Show Seleched Cel A X qw Pr REA OA n Enlarged image in the Image View with four cells selected F wu TE Tepe Teed DE TogdetsgGeaph w Pus ab rid ibe rar lates a TARI MGM Charter 10 Taie Hara Deta ar Hare ct Cal Herrtakrad nba Psa LE ea LE asa Graph View 340 oO dt s E displaying the m Fi amplification plots E ean Pa qm dl and dye intensities m4 az Pa of the four selected a7 SEN 3 Fal cells z mal umm F ae Fi d E M 1 a T 19 Ve 18 Ce ae EL E Ei AU o Raw data amplification plots and dye intensities are displayed in the Graph View when cells are selected in the
94. eal Time PCR Analysis Software User Guide Using the Dispense Map Editor These graphics show custom loading and how it looks as you proceed Dzperua Mapua E MM Tage Bauma Wel CECE Fa o MN C ee ome S NL H Al Dasergieri i l l 7 Darpersang nzfames cen Hes lel nce ol gm a aa en uni SESS plate to AL AI Race Detector T 1 1 e iren Egg X d Fe F qd te n pt dis xs 3B ik e E P zx a 25 E1 ea D J _ i _ Hn 3 h H W Gil an E Ht a zy x 5 zm zs E a E amp E be 2E PF za de o LEI t E 2 i x Reming Contr n z k Pa fegn larg amp t 3m ron E ng Target firs i 1 id AT 5 2 i amp 1 a TI I bmHusm MH e 2 tan i A oo a pe d 1 amp 8 i Ll 3n pa i af zu e LJ I x wu m n mni e n LA J a a i Rui eal uen 4E IE D uw AB L I l an L I i amp c L J a lb 4 Laper gus schaue hope Hae ier cada ol SIG plate to 4E 4B Kaze Detector ir sa IE zu F a n H x E Ax AL 5 i ii tid ir 20 a E bore nnn Fs P Begn Eden n Hop Eirg 4 AT mue j a amp aO i PEMOPNM Sk X n puni ijen i x n x 8 a i5 w y rY TY r fi a i i i oJ k i j v P 7 L To Y i i 7 Review the loading patte
95. eee eG 163 Reverse Transcription Specific Target Amplification RT STA 163 Preparing 10A Fea E adc ex oe ded do CERRO OR eor e De i 165 Preparing Sample Pre Mix and Samples llle 165 Priming and Loading the Dynamic Array IFC 1 0 ee 166 Using the Data Collection Software e m nn 166 Fluidigm Real Time PCR Analysis Software User Guide Introduction This protocol is intended to be used for fast gene expression analysis of single cells using TagMan Gene Expression Assays on the BioMark HD System The protocol includes three sections 1 single cell sorting 2 reverse transcription and specific target amplification RT STA and 3 real time PCR on either 48 48 or 96 96 Dynamic Array integrated fluidic circuits IFCs Individual cells are sorted by Fluorescence Activated Cell Sorting FACS into a 96 well PCR plate RT STA is carried out on a 96 well thermal cycler using the CellsDirect One Step qRT PCR kit and gene specific primers included in the TaqMan assays This reaction generates sufficient template cDNA for TaqMan real time analysis on Dynamic Array IFCs of hundreds of genes from hundreds of single cells in parallel Fast real time PCR for gene expression analysis requires a BioMark HD System which includes a thermal cycler with fast cycling capabilities Quanta PerfeCTa qPCR Fast Mix from Quanta Biosciences also available from VWR is used in combination with TaqMan Gene Expression As
96. elow Any modifications are rendered immediately in the heat map The changes will be saved with the chip run Fluidigm Real Time PCR Analysis 1 10 x File Edit View Report Tools Help DoWuo El 9 Chip Run Summary 1361365177 o Analysis Views T Sample Setup i B Sec Stn C Dee 00 Ji 10 13 4 14 3 Task Analyze Click the Analyze button To analyze a chip run file for the first time or After analysis parameter is changed w r2 ro ro ro ro ro ro r2 o e ceo ro M o n e mlofololnm lm lnm to to to 4 Analysis Settings qPCR MCA T Analyze a chip run using one of the Ct threshold methods below Amplification If you choose a Manual method you can enter the desired Ct threshold s Quality Threshold 0 65 Baseline Correction Linear Ct Threshold Method Auto Global 81 Temperature Eva Green at 20 C Ready From within the B dialog customize the heat map layout by changing the way rows and columns are displayed Use the graphic below as a guide 90 Fluidigm Real Time PCR Analysis Software User Guide Layout View Ln Sort names in ascending order Row Order Sample s aj di vu d i Sort names in descending order Visible Hidden iis Sort numbers in ascending order LE Sort indices in descending order j Move selection up one position Li Move
97. erence 00001 Test v v 548 407 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 408 reference to reference 00001 Unknown 1 0000 reference 00001 Test v wv 548 A09 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v E 548 410 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 A11 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 412 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v jd 548 A13 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v g 548 414 reference to reference 00001 Unknown 1 0000 reference 00001 Test v wv 548 A15 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v i 548 416 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 A17 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v F 548 A18 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v n 548 A19 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v S4n A n reference Fo reference nnnnt Linknewn 1 nnn i reference nnnnt Test D d P d M4 4 Record 1 of 2304 Calculating Delta C Detector Values To calculate A C detector values follow the procedure described in the previous section Calculating Delta Ct Sample Values on page 105 Steps that are specific to the procedure for calculating A C detector values are described
98. es of a calibrator Name Chamber ID C value Concentration Error and Call 116 Fluidigm Real Time PCR Analysis Software User Guide DE i AX Calibration View 4 f Fam mee gt ei Show Selected Rows Detector Calibrator Name Style Calibrator Count San cena see Co Error Call F E Fansa mecmreonea sie E 516 15 70 0 00 0 06 FamGUSB Weighted Linear NN Std 6 S17 15 58 0 00 0 01 v FamHPRT Weighted Linear Std 6 518 15 75 0 00 0 09 wv FamPGK1 Weighted Linear Nds si3 13 70 0 0039 0 02 wf FVGAPDH Weighted Linear RENI f 1 f Std 5 914 13 86 0 0039 0 10 v FVGUSB Weighted Linear ay d i I N meee O53 FVHPRT Weighted Linear Cee LAE e aiii FVPGK1 Weighted Linear Std4 S10 11 82 0 0156 D 03 w EZ NRC Weighted Linear std4 511 1184 0 0156 0 03 A VicGAPDH Weighted Linear Std 4 512 11 92 0 0156 0 07 w or VicGLISB Weighted Linear Std 3 S07 9 53 0 065 0 02 wv VicHPRT Weighted Linear iV Std3 508 9 63 0 065 0 00 f VicPGK1 Weighted Linear Std 3 so9 9 42 0 065 0 97 S TA li v 4 4 4 Record 1 of 13 gt lP 4 Record 1 of 54 Ce shold f 4 sg nethod LA d grail dh Toggle Log FamGAPDH 7 8 9 zyz 3 15x45 93 R20 16 00 14 00 v 5 12 00 3 v lobal Bj 10 00 8 00 0 0010 0 1000 Select a detector and its corresponding calibrators are listed on
99. etector Name A01 Ct 14 39 a Call pass 06 S06 Tm 999 00 Click the cell to display the information on the bar also 5 Optional select a row or a column by clicking an inlet or using the right click menu as shown below Click to select a column PAY tell Ma and Click to select a row Hover cursor over Select Column cell and right click to open options menu Select Row Copy Pass Fail No Call Clear Fluidigm Real Time PCR Analysis Software User Guide 77 Viewing Chip Run Data in the Data Analysis Software 6 Optional Click the upper left corner in the heat map to select all cells Analysiz Views i B E Fae GS amp STF EvaGreenatzo cct mx EEEEEEEEFFEEEEEEEEEEEEEEEEEEEEEEEEFEEFEEEEEEEE J F EEE Eee a le EREREEEEEbE RI E E E m e Ana ly sls VIEWS e 19 Heat Map View ei SHOW ERI amp al u ajdwes OCSI Heat Map View Tool Bar Map EJ GE BS Heat Map view ei Show Selected Cell k p PE ET EN Lim Eva Green at20 C Ck 7 Layout Inlet based View m EvaGreen CE EvaGreen Inside Tm Chip based View EvaGreen Outside Tm Custom view ES Auto Ed width Height Shaw Grid Show Cell Text E Edit Auto Range Color Lookup Editor You can define a range of valid C or T values using the Color Lookup Editor Preferences 78 Fluidigm Rea
100. ferent chip types 138 e Click Next 11 Click Browse to find the thermal cycling protocol files For BioMark HD GE Fast 48x48 PCR Melt v2 pcl GE Fast 96x96 PCR Melt v2 pcl 96x96 chip 48x48 chip For BioMark GE 48x48 PCR Melt v2 pcl GE 96x96 PCR Melt v2 pcl 12 Confirm Auto Exposure is selected 13 Click Next 14 Verify the chip run information 15 Click Start Run Segment Type Temperature Duration BioMark HD BioMark C seconds Ramp Rate Ramp Rate C s C s 1 Thermal 70 2400 5 5 2 Mix 60 30 5 5 2 2 Hot Start 95 60 5 5 2 3 PCR 30 96 5 5 5 2 Cycles 60 20 5 5 2 4 Melting 60 3 1 1 Curve 60 95 1 C 3 s 19C 3 s 1 Hot Start 95 60 5 5 2 2 PCR 30 96 5 5 5 2 Cycles 60 20 5 5 2 3 Melting 60 3 1 1 Curve 60 95 1 C 3 s 1 C 3 s Table 8 Thermal cycle parameters Fluidigm Real Time PCR Analysis Software User Guide Using the Real Time PCR Analysis Parameters Using the Real Time PCR Analysis Parameters 1 ul A W N Double click the Real Time PCR Analysis software icon on the desktop to launch the software Click Open Chip Run Double click a ChipRun bml file to open it in the software Enter detector and sample information Select Analysis Views We recommend using the AutoGlobal method to set the threshold We recommend using Linear Derivative as the baseline correction method For more information about baseline corre
101. for CCVM are named and have concentration values Sample Editor Type E Sample Name My first standard Relative Conc 037 Reference Use F2 key to update and move to the I Update 8 Set up Detector plate For more detailed information see Setting up a Detector Assay Plate on page 44 9 Select Analysis Views 10 Click the Analyze button The Analysis Views item now has a plus sign in front of it 114 Fluidigm Real Time PCR Analysis Software User Guide 11 Expand the plus sign next to Analysis Views and the Calibration View option appears on the tree fl BioMark Real Time PCR Analysis Chip Explorer ES A Analysis Views Calibration View Sample Setup ES E Detector Setup Tools E E Chip Run Summary 1131055065 Help 12 Select Calibration View to launch the CCVM page CCVM Page Example Below is the CCVM page which consists of five individual panes and two tool bars Chip Run Explorer BioMark Real Time PCR Analysis File Edit View Report Tools Help Chip Explorer amp Chip Run Summary 1131055065 GE Analysis Views o Calibration View ir m Sample Setup HH Detector Setup Analyze Click the Analyze button To analyze a chip run file for the first time or After analysis parameter is changed Analysis Setting Analyze a chip run using one of the Ct threshold methods below If vou choose a Manual method y
102. ftware Requirements ee 143 Preparing the Reverse Transcription RT Reaction Assembly 143 PWC CMO PEUT 145 Preparing LOX STA Primer MIX e rne 145 Preparing STA Reaction MIX 0 a 146 STACCVOIIDO a dt dir eoo Y HE e Sa ea Aor oe a Ee Eod xw 146 Exonuclease Exo I Treatment Method leen 146 Preparing the Sample Pre Mix and Samples ls 148 Preparing the 5 uM 10X Assay MIX Ie 149 Priming the Chip and Loading Assay and Samples 150 Using the Data Collection Software nne 152 Using the Real Time PCR Analysis Parameters llle 153 Fast Gene Expression Analysis Using TaqMan Gene Expression Assays HAC OCUCEIOM RR RERTTRRT SVO at ee a te a ew She a a 01 21701077 156 Required ReagentS e Nis hh eh ees 156 Required Equipment 0 ee ee ee eee es 156 Redulleg Softwa d uuu Sou n9 Keck a Gow ae CR ode he ee 156 Specific Target Amplification STA 0 e 157 Preparing LOXASSBYS i ugs ac axe hd ye RU ae RS e CR E Y ES 157 Preparing Sample Pre Mix and Samples ee ee es 158 Priming and Loading the Dynamic Array IFC 2 00 cee ee ee 158 Using the Data Collection Software ee nne 158 Using UNG for Preventing Carryover Contamination 0000 es 159 oingle Cell Fast TaqMan Gene Expression Real Time PCR Using Dynamic Array IFCs jusgoreibiaud averting os PCI PI 162 Required Reagents
103. g samples and reagents into microwells on the chip frame the chip is placed into the IFC Controller You use a laptop computer and a software user interface to control the valves and pressure load the sample and reagent fluids Samples and reagents are automatically routed to their respective chambers for PCR amplification 18 Fluidigm Real Time PCR Analysis Software User Guide Before You Begin BioMark HD System Process Overview The simplicity of running experiments on either BioMark HD System is illustrated in the five step process below For more information see Fluidigm 48 48 Real Time PCR Workflow Quick Reference PN 68000089 and Fluidigm 96 96 Real Time PCR Workflow Quick Reference PN 68000130 1 Prime the chip 2 Add the samples and assays to the chip 3 4 Load and mix samples and assays Run your real time experiment on the BioMark HD System Before You Begin To ensure good experimental results follow the guidelines listed below Organizing Your Work Label all reagent and reaction tubes Maintain a separate DNA free laminar flow hood do not use nucleic acid Samples in this hood Use dedicated pipettes tubes and gloves for all manipulations that do not involve nucleic acid samples which never leave the DNA free Sample laminar flow hood Preventing Contamination Manipulate DNA samples under a dedicated laminar flow hood name it Sample for example Use separate dedicated pipettes
104. g the Fluidigm 20X DNA Binding Dye 2 Ina 96 well plate combine 3 3 uL of Sample Pre Mix with 2 7 uL of STA and Exo l treated sample to make a final volume of 6 uL Sample Mix solution 3 Vortex the Sample Mix solution for a minimum of 20 seconds and centrifuge for at least 30 seconds Prepared reactions can be stored at 4 9C overnight 148 Fluidigm Real Time PCR Analysis Software User Guide Exonuclease Exo Treatment Method Preparing the 5 pM 10X Assay Mix The same preparation of primers can be used for the Fluidigm 48 48 Dynamic Array IFC and the Fluidigm 96 96 Dynamic Array IFC Prepare primers as shown below 1 Combine the following Table 3 Assay Mix solutions Volume for per Inlet with Overage pL 2X Assay Loading Reagent 2 5 3 0 25 0 Fluidigm PN 85000736 yellow cap Volume for 50 pL Stock Volume for per Component Inlet uL 1X DNA Suspension Buffer Teknova 2 25 2 7 22 5 PN T0221 100 uM each of Forward and Reverse 0 25 0 3 2 5 Primer Mix 2 Vortex the Assay Mix for a minimum of 20 seconds and centrifuge for at least 30 seconds to spin down all components i K IMPORTANT Vortex thoroughly and centrifuge all sample and assay solutions before pipetting into the chip inlets Failure to do so may result in a decrease in data quality NOTE The final concentration of each primer is 5 uM in the inlet and 500 nM in the final reaction Fluidigm Real Time PCR Analysis Softw
105. g the TM RANGES amp suas dads E dx dex SR o ee Working with VieWS Ne ee Result Table VIGW zai Syme co PIC imet eod FRA Cc ce dede ded Heat Map View RQ eee mrs Grab VIEW quos eux ee NN equa na Se Inu xa did actus os em d Tine Melting GRAD xw Ne ox do EA C ead EX AS xe xx Lx EXDOPEIBG D Gb Pur a xine o RENE pud EUR D a a Re iE es b ar eet cute d Fast Gene Expression Analysis Using EvaGreen on the BioMark or BioMark HD System IDEE OU ELOD APO Lais dE ente E UP ce cb VE Xe OES dc bd RETTENS cus aud osi Cae od E qe RE Xa eed qe se deuda ReguWNeQd Reagents oaa sss he rra hr hara Redired Equipmehlt 2 4 ect ocho ew c ACE og ce eS Software Requirements nn Specific Target Amplification STA le II Exonuclease Exo I Treatment Method lene Preparing Sample Pre Mix and Samples l lene Preparing the Assay MIX rn Priming and Loading the Dynamic Array IFC 1 Using the Data Collection Software ne Using the Real Time PCR Analysis Parameters lesen Fluidigm Real Time PCR Analysis Software User Guide Appendix B Appendix C Appendix D Two Step Single Cell Gene Expression Using EvaGreen Supermix on the BioMark or BioMark HD System MEFOQUGEIONS sae Ven urbe we ot A hack Oh eed eee hae Eb d dub us d es 142 Required ReagentS a we iee eea e a e e e E a i A 142 Required EQUIDIMONE uuo did a a ah ee a ee Aa EROR S 143 So
106. gm IFCs may not be used with any non Fluidigm reader and Fluidigm readers may not be used with any chip other than Fluidigm IFCs Fluidigm IFCs are single use only and may not be reused unless otherwise specifically authorized in writing by Fluidigm All Fluidigm products are licensed to the buyer for research use only The products do not have FDA or other similar regulatory body approval The buyer may not use the Fluidigm system any component parts thereof or any other Fluidigm products in any setting requiring FDA or similar regulatory approval or exploit the products in any manner not expressly authorized in writing by Fluidigm in advance No other licenses are granted expressed or implied Please refer to the Fluidigm website at www fluidigm com for updated license terms Fluidigm the Fluidigm logo BioMark EP1 FC1 MSL NanoFlex Fluidline Access Array Dynamic Array and Digital Array are trademarks or registered trademarks of Fluidigm Corporation in the U S and or other countries Fluidigm Corporation All rights reserved Limited Use License to Perform Pre Amplification with Fluidigm Chips The purchase of Dynamic Array IFCs from Fluidigm Corporation conveys to the purchaser the limited non transferable right to perform pre amplification methods under license from Life Technologies Corporation for use with the purchased amount of this product and Fluidigm instruments No right to resell this product and no other rights such as real ti
107. h The log graph shows more detail of the same view Note the finer scale on the log graph y axis below left 94 Fluidigm Real Time PCR Analysis Software User Guide Using the Graph View Amplification Log graph off Cycle Amplification Log graph on Cycle Changing Pass Fail If the heat map reveals a problematic experiment you can manually change the call to exclude the experiment Change cells to pass or fail as appropriate After reviwing the data you can manually change the call to Pass Fail or No Call No Call indicates that the data in the chamber is determined to be invalid and should not be considered in further calculations In the example below the passing cell is manually failed Pass Fail a No Call 1 Click a cell to activate it 2 Click the Pass or the Fail icon Or click Edit Pass Fail Chamber ID 522 414 Sample Name 522 Detector Name 414 Ct 26 25 Call pass Re R bed bed eee bel ed e o be 2 R S 8 8 8 SERIEM Fluidigm Real Time PCR Analysis Software User Guide 95 Viewing Chip Run Data in the Data Analysis Software If you used two probes make sure the appropriate graph tab is active In the example below FAM MGB and VIC MGB probes each have a tab on the graph view Click the appropriate graph tab before changing the call Probe specific column heads V Bela Real Time PEEL ARA IS Sancio Hacer en ru Dr ct gl ceu m Gene Fagor Type uyr fy vam Guat
108. hip Run au A WN 52 Fluidigm Real Time PCR Analysis Software User Guide Viewing Chip Run Data in the Data Analysis Software Working with AnalysisSettings lee Re 54 Changing the Quality TRE BDOIU e koe ee dob we eek ee eee ede x ES 54 Changing the Baseline Correction 0 0c eee ee 54 Changing the Ct Threshold Method llle 55 Working with Analysis ViewS ee RR RI Ie 57 Using the Results Table lll 69 Using the Image View 4 444544 64 04 4 ORC CR C9 CECI OSD AAN OO e 4 69 Image View Tool Bal ioa osos ad ban Gee dean dows oo a NE Hee S 71 Adjusting the Size of the Location Reference Map 00005 73 Using the Heat MS oo aoe 6 ori P PK ww OT xo d XC ES 75 E e ga ea ee oe E 4 Oe oe ee es E 8 8 89 Ingo Based VIEW 2644454640 arua XX oes RQ P Y 4 Y ReaD KER 89 cnip Based VIEW auod ee a ee ose eee eee Y e RN 89 CUSTOM VIEW xou eacs c ore ARE bo eG EC EO dede e EED 90 Using the Graph VIEW 6 444 44d dx oo ACE EOS eR dee e RE dede eoo a 92 TOGO the Threshold a acea NN em xo ee EO eS aE 93 Using the Graph Edit Button COs uoce 9 dn anand va odd Deed Res 94 pies fg Apr P eae rr re eee eee are ee ee are 94 150018 LOU Grah a sco C he ook dex doo ox EO ECC YO H9 XR ee 94 Ciena Pass Fall 44 acu dedo od ob eb OE TEESE ES PR XO E 95 Using the Animate Feature llle nn 96 Selecting a Single Cell ool n KK t IH 98 Selecting More Than One Cell 2 98 Cross Highlighting an
109. hip Runs When a DNA binding dye is used for detecting PCR products the products of the reaction can be analyzed by following the run with Melt Curve Analysis MCA This chapter provides a brief overview for when you analyze a chip run that has been run with a qPCR4MCA protocol Fluidigm i Se M EE ALN ALE we 31523 Chip Run Data Collection Information Barcode 1361403034 PCR Thermal Protocol 96x96 qPCR SA BioSci 10min Hot Start 30cyc Chip Type 96 96 136x File Location D ChipRunssPCR MCA 1381403034 sChipRun bml Protocol 96 86 qPCR SA BioSci_10min Hot Start 30cyc MCA Melting Passive Reference ROX 0 Phase 60 95 Probe Type s Eva Green at 20C rt Denaturats ling Experiment Start Time 5 27 2010 11 23 50 PM Duration 01 24 35 5 5 SystemlD BIOMARKO44 Commen t aunjesaduia Analysis Views Chip run results are ready for review Sample Setup 96 Wellplate Detector Setup 96 Wellplate The protocol must have one PCR segment that is before one Melting Curve segment This applies to any real time chip type By running a Melting Curve segment after the PCR segment th e T data is generated and used to validate the C curves of the PCR segment The software detects up to two T peaks one in a user defined range and one outside it Running a Chip with a qPCR MCA Protocol When you run a chip with a qPCR and MCA protocol the T peak detected inside the T detection
110. ial phase of PCR real time quantification is a reality PCR Fundamentals To appreciate the advantages of real time PCR a short review of PCR fundamentals is in order At the start of a PCR reaction reagents are in excess both template and product are at low enough concentrations that product renaturation does not compete with primer binding and amplification proceeds at a constant exponential rate The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is variable and at the plateau phase the amplification rate drops to near zero Amplification 4th phase Plateau Phase Q 3 d phase Linear Phase E Vv y vU 2 2 phase Exponential Phase 1 phase 0 10 20 30 40 50 Real time PCR cycle The Exponential Phase To ensure accuracy and precision quantitative data is best when collected at a point in which every reaction is in the exponential phase of amplification this being the only phase in which amplification is easily reproducible 14 Fluidigm Real Time PCR Analysis Software User Guide Advantages of Real Time qPCR TaqMan Chemistry The BioMark HD System uses dual labeled probes such as TagMan probes for real time qPCR amplification Dual labeled probes are oligonucleotides that contain a fluorescent reporter dye on the 5 base and a quencher located on the 3 base When irradiated the excited fluorescent reporter dye transfers energy to the nearby quencher mole
111. idigm Real Time PCR Analysis Software User Guide Setting Up a Sample Plate Fie Edit View Report Tools Help it CH iip Ex plorer E Chip Run Summary Gene Expression E Analysis Views E ays Sample Setup au S S x 5 le M ing Vi ype RU ype NTC 6 ampie Mapping view zh SampleTest_O1 Name SampleTest 01 l Bl Detector Setup Sample Editor Type OONTC Sample Name SampleTest 01 NTC Relative Conc Name SampleTest 0 i Reference a F Task Type rite ype NTC Mame SampleTest_O1 SampleTest 01 Setup Click one of the followings New to create a sample plate Export to save a plate for reuse Import to open an existing plate C Plate Settings Source 96 wellplate Name Barcode Mapping PR D Sample Contents Passive ROX Reference Contents S Name Included i s Blank v gt Nac O NTC v Q unknown Q Reference v References Type Unknown Type Unknown Type Unknown Type B Unknown Name Name Name Name lj rConc 1 rConc 1 rConc 1 rConc 1 t Ref Ref Ref Ref F 10 Close the Sample Editor Fluidigm Real Time PCR Analysis Software User Guide 37 11 Click the Open Mapping File icon Fluidigm Real Time PCR Analysis File Edit View Report Tools Help EE E I RE E Chip Explorer B Chip Run Summary 1361440131 A Analysis
112. ilter b Enter the target value 0 8 in this example Or c Click the Field checkbox to activate the menu and select a filter 68 Fluidigm Real Time PCR Analysis Software User Guide Using the Image View Custom AutoFilter Show rows where Name equals v v Field Conc Oad Oo a rConc Mame Value v 4 Optional Continue delimiting your search by clicking And Or and then selecting filters from the menus Custom AutoFilter Show rows where Quality lis less than v Field 8 and Oor v Orea E does not equal is greater than is greater than or equal to is less than is less than or equal to v 5 Click OK Using the Image View View images from individual cycles in this window 1 Click the Results Table menu 2 Click Image View Analysis Views 3 Image view dl Show Se B Results Table E Image View E Heat Map View The default Image View opens Fluidigm Real Time PCR Analysis Software User Guide 69 Viewing Chip Run Data in the Data Analysis Software a dM Chphun uy 11 172195 4048 Ch E03 at 1mage iem ETE B TT Some kal ME Decio Setup b ick the Furisiyzm button Ta analyse a chap nan Be bar tha rit bret c silts raii parental di changed Areas eingi PCR MCA Jenae a chip nun ung cres ol diee Cr theehold Mahadi bakra lE p u choote Manual method p
113. in the Data Analysis Software 12 Click Update The changes are recorded as shown in the example below DS Driolbark Beal Time PCR inih z 13 Click the mapping icon The Open Sample Mapping File dialog opens Open Sample Mapping File Look in SamplePlate M48 Sample SB596 Left dsp M48 Sample SBS96 Right dsp My Recent Documents File name My Network Files of type Dispense Mapping files dsp Cams 14 Double click left or right mapping 15 Click Analysis Views 108 Fluidigm Real Time PCR Analysis Software User Guide Calculating Delta Ct Detector Values 16 Click Analyze A C sample values are now available in the Results Table view Analysis Views Experiment Information FAM MGB Chamber Sample FAM MGB Delta Ct Sample ID Name Type rConc Reference Mame Type Quality Threshold value Quality n 548 401 reference to reference 00001 Unknown 1 0000 reference 00001 NRC 0 92 0 91 E 548 A02 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 A03 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 A04 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v 548 405 reference to reference 00001 Unknown 1 0000 reference 00001 Test v v E 548 406 reference to reference 00001 Unknown 1 0000 ref
114. inutes The complete program is as follows 70 C for 30 minutes 25 C for 10 minutes 95 C for 1 minute followed by 35 cycles of 96 C for 5 seconds and 60 C for 20 seconds Using UNG for Preventing Carryover Contamination The Quanta PerfeCTa qPCR Fast Mix and the TagMan Fast Advanced Master Mix both contain UNG If using these master mixes the cycling program can be modified to include a UNG step to protect against carryover contamination Note the following e For the Quanta PerfeCTa qPCR Fast Mix a 2 minute incubation at 45 C is recommended by the manufacturer For the 48 48 Dynamic Array IFC this should be added at the beginning of the program For the 96 96 Dynamic Array IFC this should be added before the Hot Mix step e For the TaqMan Fast Advanced Master Mix a 2 minute incubation at 50 C is recommended This should be added at the beginning of the program for the 48 48 Dynamic Array IFC and before the Hot Mix step for the 96 96 Dynamic Array IFC The TagMan Fast Universal PCR Master Mix contains dUTP and can be modified by the addition of UNG The TaqMan GT Xpress Master Mix was designed for genotyping applications and does not contain any dUTP Fluidigm Real Time PCR Analysis Software User Guide 159 160 Fluidigm Real Time PCR Analysis Software User Guide Single Cell Fast TaqMan Gene Expression Real Time PCR Using Dynamic Array IFCs es oca 394 PE Nard eee keh E E ee oe ae 162 Cel Pie o ea 4 oe ehh eee ewe ee EAS Ee
115. is Column RO1 C36 548 Group By Box RO1 C3 548 amp Column Chooser RO1 C38 548 Best Fit RO1 C39 548 R01 C40 548 F RO1 C41 548 Best Fit all columns Rfh1 C4 S4A TIRAT 3 Drag and drop unwanted Analysis Views D ix Results Table d Show Selected Rows Experiment Information Chamber Sample ID Type Name rConc b R48 CO1 Unknown S01 R48 CO02 Unknown 501 R48 CO3 Unknown 501 R48 CO4 Unknown 501 R48 COS Unknown S01 R48 C06 Unknown 501 R48 C07 Unknown 501 R48 CO8 Unknown S01 R48 C09 Unknown 501 R48 C10 Unknown 501 R48 C11 Unknown S01 R4R C1 HInknieiuim sni Experiment Information Chamber Sample FAM MGB ct ID Type 1 Mame Type Value gt R48 CO1 Unknown 1 00 D01 Unknown R48 C02 Unknown Customization R48 CO3 Unknown Columns Bands R48 CO4 Unknown R48 COS Unknown R48 CO6 Unknown R48 CO Unknown R48 CO8 Unknown R48 C09 Unknown R48 C10 Unknown R48 C11 Unknown R4R C1 I Inlerieiar GE Ec Results Table gl Show Selected Rows rConc E v UU DOF X Customization Columns Bands Key Key column headers onto the Customization dialog FAM MGB FAM MGB ct Name Type Yalue Qua Te n 1 Tod Yom qo Customization x Columns Bands 1 00 DO 1 00 DOs 1 00 D09 1 00 D10 1 00 Dil 1 nnin1 66 Fluidigm Real Time PCR Analysis Software User Guide Working with
116. isting chip run pening an existing chi r creating a new chip run EEE Quick Tasks al Open a Chip Run Create a New Chip Run Recent Chip Runs S 413614401 31 ChipRun bml S 413613651 7 ChipRun bml A N13861402100SChipRun bml S NT131055065sChipRun bml 888822001 1 GE ChipRun bml 41130212156 ChipRiun bml 4 41131054061 GE perf ChipRun bml 2 Double click the chip run file bml extension O 1130076016 a cals 2 Data My Recent Documents File name ChipRun bml My Network Files of type bral files bral The chip run file opens 3 Click Analysis Views Fluidigm Real Time PCR Analysis Software User Guide Finding Corners Manually iFluidigm Real Time PCR Analysis B 1 101 xi File Edit View Report Tools Help Do la El 9 Chip Explorer EM Chip Run Summary 1361440131 T E Results Table e Show Selected Rows Analysis Views ger Sample Setup o zii Detector Setup Sample Type Takara ExTag Unknown 1 00 FBX032 Reference Mame Type rCone Takara ExTaq Unknown 1 00 GDF11 Test FBX032 Takara ExTaq Unknown 1 00 Trim63 Test FBX032 Takara ExTag Unknown 1 00 ACTB Test FBX032 Takara ExTaq Unknown 1 00 CTSB Test FBX032 Takara ExTaq Unknown 1 00 FoxM1 Test FBX032 res Takara ExTaq Unknown 1 00 KRT18 Test FBX032 Takara
117. l Show Selected Rows a EI INA l name l Type nt gt id onc 1 00 fej em e 501 Unknown 1 00 D47 FAM MGB Name rCone Click plus or minus to expand collapse the windows Fluidigm Real Time PCR Analysis Software User Guide 61 Viewing Chip Run Data in the Data Analysis Software 62 5 Drag and drop one header element over another as shown below to change places hierarchy The hierarchy dictates how the data displays as you expand windows In this example the ID header is dragged over the Type header and then dropped They exchange places as a result E Analysis Views FE Results Table d Show Selected Rows Name 7 l us Type uli ID Experiment Information Chamber Sample ID i Name 532 Type Name 2 Type Unknown gt E ID R17 CO1 R17 532 ID R17 CO2 ID R17 CO3 Unknown Experiment Information Chamber ID i Name 532 iD R17 C01 zi Type Unknown Sample Name Type R17 532 Unknown ID R17 CO2 ID R17 C03 Fluidigm Real Time PCR Analysis Software User Guide Ungrouping One Header 1 2 o amp Chip Run Gene Expression A Analysis Views E m Sample Setup Sample Mapping View Bl Detector Setup Task nx Ungrouping All Headers 1 2 Click Clear Grouping 5 Chip Run Gene Expression A Analysis Views eur Sample
118. l Time PCR Analysis Software User Guide Using the Heat Map 1 Click the Color Lookup Editor button I9 2 Choose Ct YellowToBlue or Tm YellowToBlue 3 Click Edit Color Lookup Editor Ct YellowT oBlue Ct YellowT oBlue Ct YellowT oBlue_T est Tm YellowT oBlue g Data Range Number of color segments Minimum Value 60 00 Maximum Value 9500 S Spectrum Editor Color Spectrum Eva Green at 20 C Ct Color Interpolation Method RGB Spectrum definition colors Invalid data color Minimum 33 67 Maximum 4 Choose RGB red green blue or HSL hue saturation lightness S Spectrum Editor Spectrum Editor Color Spectrum Eva Green ati Color Spectrum Eva Green at 29 Color Interpolation Method eim Color Interpolation Method C Pe Spectrum definition colors S SI Spectrum definition colors b Invalid data color mm Invalid data color Minimum Minimum 33 33 67 67 Maximum Maximum 0200 00 00 0 00 OCOUNNNNNNNNNNNNEEENEEEEEEEMMMENN HSL 5 Optional Change the percentage increments between colors by changing the number a Click Edit b Change the value from 1 to 20 c Click OK Fluidigm Real Time PCR Analysis Software User Guide 79 Viewing Chip Run Data in the Data Analysis Software S Spectrum Editor Color Spectrum Eva Green at 20 C Ct Color Interpolation Method RGB v Spectrum definition colors cs gG Invalid data color Minimum
119. le the text view on and off Zoom Increase or decrease the image view size by multi clicking the magnifying glass icons and 88 Fluidigm Real Time PCR Analysis Software User Guide Layout View Layout View Toggle between inlet based chip based and custom views In the heat map Layouk Inlet based View Inlet based View Chip based View BRI Custom View he ITE Mel bb bbb debe e el eb ell A nu ICI mp Le Lee be be be Le bere Le Le Le Le Le Lee Le Le rae 4 h hel Lakebebebebealelet c c be be b b be b EtbEEREEERE Pde E E NEL itt Inlet Based View The inlet based view shows the cell in the same numbered sequence as the inlets on the chip as shown below Chamber ID 575 402 m Toe Sample Mame 515 3 LM E 24 1 Detector Mame A2 cR ERSS Ct 13 13 CERERE Call pass Tm 999 00 Chip Based View The chip based view shows a sequence of numbers assigned to chambers on a chip counting from top left corner to the right and then top to bottom E Detector 60 60 OL Ob LU FU cuw zn EOw En For FO sor so sor so eor en sor s0 Fluidigm Real Time PCR Analysis Software User Guide 89 Viewing Chip Run Data in the Data Analysis Software Custom View The Custom View selection adds two buttons to the tool bar one to modify the column layout and one to modify the row format Both function in the same manner see detailed description b
120. lit screen GE Using the Results Table To access the Results Table 1 Click Analysis Views 2 Select Results Table if it is on a different view File Edit View Report Tools Help O d O O rt L RA Analysis Views EL 7 sampe a Detector Setup In the Results Table view right click a column header to Fluidigm Real Time PCR Analysis Software User Guide 57 Viewing Chip Run Data in the Data Analysis Software e Adjust columns see page 58 e Group columns see page 59 e Sort columns see page 65 e Column Chooser see page 66 e Customize search filters see page 68 3D x Results Table dal Show Selected Rows Experiment Information FAM MGB Chamber Sample FAM MGB ct ID Name Type rConc Mame Type Value Cialibs Call Thresh gt 548 A01 Unknown 1 0000 NRC el Sort Ascending 548 A02 Unknown 1 0000 Test 4 Sort Descending 548 A03 Unknown 1 0000 Test 548 A04 Unknown 1 0000 Test Group By This Column 548 405 Unknown 1 0000 Test cm Group By Box 548 AD6 Unknown 1 0000 Test Bl Column Chooser 548 A07 Unknown 1 0000 Test Best Fit 548 408 Unknown 1 0000 Test 548 409 Unknown 1 0000 Test 548 410 Unknown 1 0000 Test Best Fit all columns S4R A11 I Inkrieian 1 nnnn Test In this example right click the Value column header to open the options menu Resizing Columns Using the Cursor e Position the cursor on a column edge When the cursor changes to a double arrow hold a
121. m 2 MaoF Leung W Y and Xin X 2007 Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications BMC Biotechnology 7 76 doi 10 1186 1472 6750 7 76 Required Reagents Stored at 20 C e Exonuclease New England BioLabs PN M0293L SsoFast EvaGreen Supermix with Low ROX Bio Rad Laboratories PN 172 5211 Stored at 4 C e 2X TagMan PreAmp Master Mix Applied Biosystems PN 4391128 e 20X DNA Binding Dye Sample Loading Reagent Fluidigm PN 100 3738 e 2X Assay Loading Reagent Fluidigm PN 85000736 130 Fluidigm Real Time PCR Analysis Software User Guide Specific Target Amplification STA e 100 uM each Forward and Reverse Primer Stock Mixture for each assay of interest Stored at Room Temperature e TE Buffer 10 mM Tris pH 8 0 1 0 mM EDTA TEKnova PN T0224 e PCR Certified Water TEKnova PN W3330 Required Equipment e BioMark or BioMark HD System e IFC Controller MX for the 48 48 Dynamic Array IFC or HX for the 96 96 Dynamic Array IFC e Standard 96 well Thermal Cycler Software Requirements Fluidigm Real Time PCR Analysis Software v 3 0 2 or higher and BioMark HD Data Collection Software v 3 1 2 or higher is required for this protocol Specific Target Amplification STA We recommend using DELTAgene Assays from Fluidigm These assays are provided as a Forward and Reverse primer mix with each primer at a concentration of 100 uM primer an
122. me 6 Click Update Sample Editor Type Reference Sample Name Reference 00001 Relative Conc Reference The Sample Setup reflects the change Fluidigm Real Time PCR Analysis Software User Guide 105 Viewing Chip Run Data in the Data Analysis Software EA BioMark Real Time PCR Analysis File Edit view Report Tools Help 5 H Chip Run Summary 1130106002 3 3 Editor A Analysis Views r _ m Sample Setup is Sample Mapping View SELELELLE i i Reference Reference 00001 Reference 00001 Reference 00001 1 ET Detector Setup EZ Detector Mapping View Task ax Setup Click one of the following New to create a sample plate Export to save a plate for reuse Import to open an existing plate Plate Settings Source 96 Wellplate Name Barcode Mapping 48 48 5 ample SBS96 Le Sample Contents Passive ROX Reference Contents S Name Included Blank Nac NTC Unknown Refefence References Reference 00001 106 Fluidigm Real Time PCR Analysis Software User Guide Calculating Delta Ct Sample Values 7 Select all cells that you want to reference Typically you select all the cells except for the three reference cells as in the example below Type LE Ew J Type F i Type tom Name I rConc Name rConc LIEB rcConc 4 D Referenc
123. me PCR Analysis Software User Guide 49 Converting a Chip Run to a More Samples Run A More Samples run requires its own sample and assay setup and Fluidigm provides the necessary Microsoft Excel setup templates for you Follow these steps to convert a chip run to a More Samples run e Step 1 Set up your samples Step 2 Set up your assays e Step 3 Import the samples and assays to the Analysis software Step 1 Set Up Samples 1 Goto C Program Files Fluidigm BioMarkDataAnalysis ApplicationData FileFormats 2 Open SamplePlateDefinitionForMoreS 3 Click Options to enable Active X if prompted H J K L Number of Tags 12 TagiD Oo Sample Plate 1 a a a aa pee E l ee ee eee ee 14 A 2 Clear Definition Create Plate CSW File 3 aN k Annmmorga 4 If prompted select Enable this content in the Microsoft Office Security Options dialog 5 Click OK 6 Edit the Microsoft Excel template to match your experiment 7 Click Create Plate CSV File Security Warning Some active content has been disabled Options 013 she A B C D E E J K L M 1 Number of Tags 2 Clear Definition 2 Create Plate CSV File 3 12 TaglD Sample Plate 1 EN a A 14 A 15 ES 50 Fluidigm Real Time PCR Analysis Software User Guide Converting a Chip Run to a More Samples Run 8 Open the new CSV file tab and double check your annotations a gem ona uet uer ee e were
124. me PCR methods apparatus reagents or software to perform digital PCR methods are conveyed by Life Technologies Corporation expressly by implication or by estoppel For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 For information about the scope of the above identified Fields please contact labellicenseinformation fluidigm com Fluidigm Product Patent Notice Fluidigm products including IFCs integrated fluidic circuits microfluidic chips with or without a carrier such as Access Array IFCs Dynamic Array IFCs and Digital Array IFCs the IFC controller FC1 Cycler and the Fluidigm system BioMark System EP1 System readers thermal cycler etc and methods for reading and controlling the Access Array IFCs Dynamic Array IFCs and Digital Array IFCs and or their use and manufacture may be covered by one or more of the following patents owned by Fluidigm Corporation and or sold under license from California Institute of Technology and other entities U S Patent Nos 6 408 878 6 645 432 6 719 868 6 793 753 6 929 030 7 195 670 7 216 671 7 307 802 7 323 143 7 476 363 7 479 186 7 494 555 7 588 672 7 601 270 7 604 965 7 666 361 7 691 333 7 704 735 7 749 737 7 766 055 7 815 868 7 837 946 7 867 454 7 867 763 7 906 072 8 048 378 EP Patent Nos 1065378 1194693 1195523 and 1345551 and additional issued and
125. nd drag the column bigger or smaller Or e Double click the column edge to adjust the column to precisely fit the contents Using Best Fit e One column right click a column header and select Best Fit The column automatically adjusts to precisely fit the contents All columns right click a column header and select Best Fit all columns to adjust all columns to precisely fit the contents 1alys i Y i eus GE Ex Results Table Experiment Information Cham Sample 51 Sort Ascending R48 C47 R48 C48 R47 CO1 SampleTest_O1 own Z Sort Descending Lon R47 CO2 SampleTest 01 Group By This Column R47 CO3 SampleTest O1 cm Group By Box R47 CO4 SampleTest O1 E Coli Chooser R47 CO5 SampleTest 01 R47 CO6 i ENT R47 C07 SampleTest 01 X R47 C08 SampleTest_01 Best Fit all columns R47 CO9 SampleTest 01 TAT R47 C10 SamoleTest 01 NAC 58 Fluidigm Real Time PCR Analysis Software User Guide Working with Analysis Views Grouping Two or More Columns 1 2 Right click on any column header Click Group by Box Analysis Views 3D A Results Table ol Show Selected Rows Experiment Information Chamber Sample FAM MGB ID Name x Ep Name R48 C02 s01 Al Sort Descending 1 00 D02 R48 CO3 S01 1 00 D03 R48 C04 501 Group By This Column 1 00 D04 R48 C05 501 1 00 D05 R48 C06 ET A Column Chooser 100 D06 R48 c07 5
126. ng to the table below Component Volume pL 48 Samples with 96 Samples with Overage pL Overage pL 10X SuperScript Enzyme Mix 0 15 90 0 18 0 Table 2 RT Mix Solution 2 Aliquot 1 uL into each of the wells and centrifuge briefly at 4 9C Fluidigm Real Time PCR Analysis Software User Guide Preparing 10X STA Primer Mix RT Cycling Follow the thermal cycling conditions below on a standard thermal cycler Condition Reverse Transcription 1 Centrifuge and store the first strand cDNA samples at 20 C or proceed directly to PCR Preparing 10X STA Primer Mix 1 Werecommend using DELTAgene Assays from Fluidigm These assays come as a Forward and Reverse primer mix with each primer at a concentration of 100 uM primer and Reverse primer for each assay so that the concentration of each need Q NOTE If you obtain primers from another source combine the Forward primer is 100 uM Proceed to step 2 2 Ina DNA free hood combine equal volumes of each 100 uM primer pair 3 Dilute using 1X DNA Suspension Buffer so that each primer is at a final concentration of 500 nM This mix represents a 10X concentration of pooled STA Primer Mix 4 Vortex for 20 seconds and centrifuge for 30 seconds to spin down all components 5 Store 10X STA Primer Mix at 42C for repeated usage up to six months or store at 202C for long term storage 48 Primer Pairs EXAMPLE Volume pL 1 uL each primer pair 100 uM each 1 uL x 48 2
127. nstant correction shown as black fill Amplification ARN Linear baseline correction 1 4 POSSTOLST3 18 139 22 25 28 31 34 3 40 Cycle Baselines are flattened in the Linear correction Changing the C Threshold Method To change the quality threshold 1 Click Analysis Views 2 Under Analysis Settings select a different option in the C Threshold Method field Fluidigm Real Time PCR Analysis Software User Guide 55 Viewing Chip Run Data in the Data Analysis Software Auto Global Automatically calculates a threshold that is applied to the entire chip Auto Detectors Independently calculates a threshold for each detector on a chip You must enter a unique detector name in the Detector Editor during detector setup User Global Allows you to manually adjust the threshold when searching for the C rise in slope The value is applied to all the detectors Analysis Settings gPER Analyze a chip run using one of the Ct threshold methods below IF you choose a Manual method vau can enter the desired Ct threshold s Quality Threshold 0 65 Baseline Correction Linear Ct Threshold Method Threshold FAM MGB I 0 1 User Detectors Allows for tighter control when searching for the C curve s rise in the slope You can individually set the threshold for each detector on the Ct Thresholds tab M Analysis Settings qPCR Ct Thresholds Thresholds Detector
128. o N 0 00 V o 61 71 81 91 Temperature Fluidigm Real Time PCR Analysis Software User Guide 127 qPCR Melting Curve Analysis A black vertical line represents a T peak detected outside the T detection range Melting 61 71 81 91 Temperature Exporting Data CSV Table data includes the T columns from the Table View CSV Heat Map data includes visible data in the heat map C Inside T Outside Ta 128 Fluidigm Real Time PCR Analysis Software User Guide Fast Gene Expression Analysis Using EvaGreen on the BioMark or BioMark HD System uice go o P E E E eo E eee Ae ee E 130 Specific Target Amplification STA 0 00 cee ee nn 131 Exonuclease Exo I Treatment Method 0 00 eee ees 133 Preparing Sample Pre Mix and Samples 1 0 0 eee ee es 134 Preparing the Assay MIX 0 ee 135 Priming and Loading the Dynamic Array IFC 2 0 135 Using the Data Collection Software e m nn 137 Using the Real Time PCR Analysis Parameters onanan 000 a a ee ees 139 Fluidigm Real Time PCR Analysis Software User Guide 129 Introduction The use of DNA binding dyes for gene expression analysis is a lower cost alternative to the use of labeled probes The method Is sensitive and when coupled with melt curve analysis the specificity of the primers can be confirmed For this protocol we are recommending the use of EvaGreen dye which has several advantages over SYBR Green 1 2
129. on reaction products and dilution of final reaction products 132 Fluidigm Real Time PCR Analysis Software User Guide Exonuclease Exo Treatment Method Exonuclease Exo I Treatment Method For best results we recommend using a cleanup step to remove unincorporated primers This can be done with Exonuclease E coli 1 Dilute the Exonuclease to 4U uL as shown Per 5 uL 48 Samples with 96 Samples with me e M fe Exonuclease Reaction Buffer Exonuclease at 20 units uL Total Volume Table 3 Exo1 reaction solution 2 Add 2 uL of diluted Exo at 4 U uL to each 5 uL STA reaction vortex centrifuge and place in a thermal cycler Condition Digest Inactivate Hold mm fore e ee Time 30 minutes 15 minutes Table 4 Thermal cycle conditions 3 Dilute the final products to an appropriate concentration for testing The minimum amount of dilution that should be used is 5 fold but if the C values are consistently below 6 for some of the assays this may need to be increased to 10 fold or 20 fold Use TE Buffer TEKnova PN T0224 to dilute the products as shown below Volume to Add Volume of STA 5 fold dilution 10 fold dilution 20 fold dilution Reaction Exonuclease 7 uL 18 uL 43 uL 93 uL Table5 Dilution table e Store diluted STA products at 20 C or use immediately for on chip PCR NOTE For larger volume STA reactions adjust the amounts of materials proportionally
130. ontacting Fluidigm For Technical Support Email United States and countries not in Europe or Asia TechSupport fluidigm com Europe TechSupportEurope fluidigm com Asia TechSupportAsia fluidigm com Phone United States 1 866 FLUIDLINE 1 866 358 4354 Outside the United States 650 266 6100 On the Internet www fluidigm com support Fluidigm Corporation 7000 Shoreline Court Suite 100 South San Francisco CA 94080 PN 68000088 Rev G1 Contents Preface Chapter 1 Chapter 2 Fluidigm Real Time PCR Analysis Software User Guide About this User Guide How to Use This GUIGG as nego dedo m ea eee bh bs ee SERS 11 Document Conventions rss 11 Related Documents a rss 12 BioMark System Pea HVS OP R C LTTITUTTTITIBMPIITT 14 Advantages of Real Time qPCR ouaaa s 14 PCR Fundamentals 4 a ox RON oy ee ERO Sq hae ee eee 14 The Exbonentclal PIiasG amp ok ancy ie d OA Cet awe RD C8 ee WE CREE es 14 Advantages of Real Time qPCR TagMan Chemistry 00 0 15 BioMark HD System for Genetic Analysis 0 00 eee ee 16 High End Detection Optics ssess QU 16 The BioMark HD System Components 0000 rne 17 Dynamic Array IFC Components ee 17 48 48 Dynamic Array IFC for Real Time Quantitative PCR 17 96 96 Dynamic Array IFC for Real Time Quantitative PCR 18 BioMark HD System Process Overview ns 19 BEFORE YOU BEGIN iii ieee aeni ARN e
131. ou can anbar ie dermed C1 thresholds Hualdy Threshold LJ Kassie Conrechory I s Oi Tiveshold Methot Auto Bba E i5 M Crp Bun Same 1107 848 Cho 1 FIT a Tmp iep Lusscha Seb IMPORTANT An image displays only after you have selected a dye and a cycle number A representation of the chip displays in the Image View 70 Fluidigm Real Time PCR Analysis Software User Guide Using the Image View E7 BioMark Real Time PCR Analysis File Edit View Report Tools Help 3 2 bed EL Chip Run Summary 1130114010 GE Image view Gel Show Selected Cell A Analysis Views p z TET In A 1009 a Fit zal al G Rox B M Contrast M IES Qi Sample Setup A B Detector Setup Tasi Prid Analyze i a ka uEXxxiVWRAXXERXLLTLEZEXEZIYELTYY Click the Analyze button m m wmm NR AN M MR M M M A MN M M m M M M M n To analyze a chip run file for the first time or After analysis parameter is changed Analysis Setting Analyze a chip run using one of the Ct threshold methods below If you choose a Manual method you can enter the desired Ct threshold s Quality Threshold 0 65 Baseline Correction Linear v Ct Threshold Method Auto Globa 5 Optional Click the double arrow to expand the image Image View Tool Bar Elements of the Image View tool bar are shown below Expand Image button Click to enlarge to 100 Displays lo
132. ou can enter the desired Ct threshold s Quality Threshold 0 65 Baseline Correction Linear v Ct Threshold Method User Data Global w Threshold FAM MGB 0 03 Threshold VIC MGB 0 03 Analysis Task Pane Name Primary View Tool Bar FamGUSB Weighted Linear FamPGK1 Weighted Linear FamGAPDH Weighted Linear NRC Weighted Linear VicGUSB Nu Weighted Linear VicPGK1 Weighted Linear VicGAPDH Weighted Linear VicHPRT Weighted Linear FVGAPDH Weighted Linear FYPGK1 Weighted Linear FYGUSB Weighted Linear FVHPRT Weighted Linear Record 4 of 13 gt kp Pass grail SA Toggle Log FamHPRT 10 11 12 zyz 3 32x 5 12 R 0 15 00 12 00 o 9 00 6 00 0 0010 0 1000 Graph Area Detector Table Calibrator Table Std 5 Std 4 Std 4 Std 4 Std 3 Std 3 Std 3 S16 15 04 S17 15 17 518 15 08 S13 12 96 0 00 0 00 0 00 0 0039 Error 0 00 0 00 0 00 0 02 13 06 0 0039 0 00 510 11 21 S11 11 26 S12 11 26 S07 9 16 508 9 03 509 9 09 0 0156 0 0156 0 0156 0 065 0 065 0 065 13 Sas Sie 19 12 0059 O00 S15 0 00 0 01 0 01 0 00 0 00 0 00 IE Record 1 of 54 e Secondary View Tool Bar Fluidigm Real Time PCR Analysis Software User Guide v EV v NAIA Weighted linear is a method to calculate calibration curve 115 Viewing
133. press Master Mix Applied Biosystems PN 4401892 or TaqMan Fast Advanced Master Mix Applied Biosystems PN 4444557 These volumes include some overage to account for pipetting error 2 Ina DNA free hood combine the two Sample Pre Mix components in a 1 5 mL sterile tube enough volume to fill an entire chip Aliquot 3 3 uL of the Sample Pre Mix for each sample 3 Remove the aliquots from the DNA free hood and add 2 7 uL of cDNA to each to make a total volume of 6 uL in each aliquot Priming and Loading the Dynamic Array IFC For instructions on loading the 48 48 Dynamic Array IFC see Fluidigm 48 48 Fast Real Time PCR Workflow Quick Reference PN 100 2637 For instructions on loading the 96 96 Dynamic Array IFC see the Fluidigm 96 96 Real Time PCR Workflow Quick Reference PN 68000130 Using the Data Collection Software The protocols used for data collection are fast protocols 48 48 Select GE 48X48 Fast v1 pcl in the GE folder This cycling protocol is 95 C for 1 minute followed by 35 cycles of 96 C for 5 seconds and 60 C for 20 seconds This protocol takes approximately 26 minutes 158 Fluidigm Real Time PCR Analysis Software User Guide Using UNG for Preventing Carryover Contamination e 96 96 Select GE 96X96 Fast v1 pcl in the GE folder The cycling protocol portion of this is the same as for the 48 48 but also includes the Hot Mix protocol for the 96 96 Dynamic Array IFC The total program runs approximately 66 m
134. pression Assays es PET Specific Target Amplification STA 0 0 00 cee et ee n Preparing 10X ASSAYS iis ead ace ee ee ee e s Preparing Sample Pre Mix and Samples ln Priming and Loading the Dynamic Array IFC 1 ee Using the Data Collection Software enn Using UNG for Preventing Carryover Contamination sees Fluidigm Real Time PCR Analysis Software User Guide 155 Introduction This protocol is intended to be used for fast gene expression analysis on the BioMark HD System using TagMan Gene Expression Assays The protocol is suitable for use with either 48 48 or 96 96 Dynamic Array IFCs and appropriate cycling protocols are provided for each chip type This protocol requires a BioMark HD System which includes a thermal cycler with fast cycling capabilities The protocol also requires the use of a master mix that does not require a long hot start and that works well with the shortened cycling times Three master mixes that we have found to work well are Quanta PerfeCTa qPCR Fast Mix from Quanta Biosciences also available from VWR TaqMan Fast Universal Master Mix from Applied Biosystems and TagMan GTXpress Master Mix from Applied Biosystems In preliminary testing the new TaqMan Fast Advanced Master Mix from Applied Biosystems also appears to be suitable for use with this protocol Although the four master mixes above are recommended because they work well for fast gene expression analysis
135. provides reaction densities far beyond what is possible with microtiter plates and significantly reduces the number of liquid handling steps and the volume per reaction High End Detection Optics 16 The BioMark HD System includes a high resolution CCD camera that covers 30mm by 30mm an area sufficiently large to simultaneously image all reactions in Dynamic Array IFCs The BioMark HD System optics and analysis software is available for different applications which are compatible with a variety of Fluidigm chip families for TagMans chemistry The System s computer controlled chip tray automatically loads the chip into the instrument for ease of use A barcode reader tracks experiments reducing the chance of errors Fluidigm Real Time PCR Analysis Software User Guide The BioMark HD System Components The BioMark HD System includes an internal thermal cycler flat panel monitor keyboard and mouse The IFC Controller single bay The MX model primes and loads 48 48 chips while the HX model primes and loads the 96 96 chips Dynamic Array IFC Components Although chip architecture varies the essential components common to all are highlighted in the graphic below For more information see the appendices 48 48 Dynamic Array IFC for Real Time Quantitative PCR The 48 48 Dynamic Array IFC is a matrix of channels chambers and integrated valves finely patterned into layers of silicone Valves within the array partition 48 samples
136. range validates the amplification curve of the PCR cycle If no Tm peak is detected then any amplification that may exist is not considered valid and the quality score of the C is set to zero 0 making the chamber Fail You analyze data by clicking the Analyze button The T Ranges The T peak detection range is set for each detector By default each detector range is the temperature range of the protocol 124 Fluidigm Real Time PCR Analysis Software User Guide To identify the temperature range in which you expect to see a T peak select a region of the temperature range A T peak in range validates the C curve and a T4 peak outside of range invalidates it failing the chamber The MCA tab allows you change these parameters e Peak Sensitivity adjust how sensitive the algorithm is for detecting a peak with 1 being the least sensitive and 10 being the most sensitive e Peak Ratio Threshold determine if a peak outside of range should cause the chamber to fail when multiple peaks are detected one in range and one out of range T Ranges adjust the detection range for each detector qPCR MCA Peak Sensitivity x l Peak Ratio Threshold og E Tm Ranges Detector Ld Eva Green at 20 C AED Eva Green at z0 cC 3 v Eva Green at 20 C AED amp 0 95 Eva Green at 20 C AED 60 95 Viewing the T Ranges Toggle the Threshold button to display the T detection range light blu
137. rn you have recorded by clicking the green arrow button in the playback control pane Recording Control Stop Editing Playback Control DIO Fluidigm Real Time PCR Analysis Software User Guide 43 Setting up a Detector Assay Plate Use this table as a guide when annotating detectors assays BI Test Experiment reagents Reference A control or reference gene Bl NRC No Reagent Control negative control using only buffer no primers probes detectors To Set Up the Detector 1 Click Detector Setup 2 Click New 1 X Detector Setup g Chip Run Summary 1130114010 3 Choose the appropriate Container type and Container format 4 Click OK to open the Detector Plate screen OPTIONAL Double click between columns to expand 44 Fluidigm Real Time PCR Analysis Software User Guide Setting up a Detector Assay Plate 5 Select cells by performing one of the following e Click and hold while dragging your cursor through cells e Click the upper left corner to select all the cells e Click individual cells while pressing the CTRL key Fluidigm Real Time PCR Analysis Software User Guide 45 Detector Setup Name Ref Type Name Ref I Type Type Name Name COR Ref Type est Type j Name Name Detector Setup Type elu com Test Type Name Ref Type Name Ref Type Name Click the upper left corner to select all cells
138. s the example below illustrates l Enter a value 2 Click OK Fluidigm Real Time PCR Analysis Software User Guide Using the Heat Map S Color Lookup Editor Color Scheme E v c5 Data Range Number of color segments Minimum Value Maximum Value When you click OK the heat map and the legend reflect the change also Using Auto Range Auto range allows you to eliminate a percentage of the upper and lower ranges of all valid C or T values 1 Click Auto Range S Color Lookup Editor Color Scheme Ct YellowT oBlue v Lea Data Range Number of color segments 30 Minimum Yalue 1 00 Maximum Value 3800 2 Change the values In the example below the minimum and maximum values have been changed to 3 0 Therefore after discarding the lower and upper 3 of valid values you are left with a range of 11 86 to 28 18 Auto Select Range Values The auta range selection will select a range of data values based upon the data currently available You may specify a percentage of the valid values to be discarded for bath the minimum the masium value calculations Number of valid values 7395 Minimum value after discarding lower Z 11 86 Maximum value after discarding upper Z 20 18 This range is represented in the Color Lookup Editor illustrated below Note the eliminated values from 1 to 11 86 and 28 18 to 35 are now gray areas Fluidigm Real Time PCR Analysis Software User
139. says from Life Technologies Corporation for real time PCR analysis This protocol has been verified on both 48 48 and 96 96 Dynamic Array IFCs Required Reagents e CellsDirect One Step qRT PCR Kit Invitrogen catalog numbers 11753 100 and 11753 500 SUPERase In Ambion PN AM2694 e DNA Suspension Buffer 10 mM Tris pH 8 0 0 1 mM EDTA TEKnova PN T0221 e 20X TaqMan Gene Expression Assays Applied Biosystems e 2X Assay Loading Reagent Fluidigm PN 85000736 e Quanta PerfeCTa qPCR Fast Mix low ROX Quanta Biosciences PN 95078 012 or VWR PN 1014190 220 e 20X GE Sample Loading Reagent Fluidigm PN 85000746 e PCR certified water TEKnova PN W3330 Required Equipment 162 e FACS instrument e Standard 96 well Thermal Cycler e 96 well PCR plates that are compatible with the FACS instrument and thermal cycler e Adhesive plate seals ABI PN 4311971 e IFC Controller MX for the 48 48 Dynamic Array IFC or HX for the 96 96 Dynamic Array IFC e BioMark HD System Fluidigm Real Time PCR Analysis Software User Guide Cell Sorting Procedure Software Requirements Fluidigm Real Time PCR Analysis Software v 3 0 2 or higher and BioMark HD Data Collection Software v 3 0 2 or higher is required for this protocol Cell Sorting Procedure 1 For each 96 well PCR plate prepare 2X reaction reagent by mixing 580 uL of the CellsDirect 2X Reaction Mix with 11 6 uL of Ambion SUPERase In 2 Pipette 5 1 uL of the
140. selection down one position Delete Tone bly Delete Toggle Visiblity Toggle Replicates Move selection to Hidden panel 3 Move all to Hidden panel When enabled will hide or show all rows and columns that share the same Move selection to Visible panel name as the selection 4 Move all to Visible panel Click and drag the cursor to select a group To change row order 1 Click the row order button The Row Order Sample dialog opens 2 Group reorder and hide rows and columns as needed Fluidigm Real Time PCR Analysis Software User Guide 91 Viewing Chip Run Data in the Data Analysis Software Using the Graph View The graph view displays the curve data and information about the current selection of chambers The secondary tool bar allows you to change the display of the data Chip Explorer Analysis Views E Chip Run Summary 1361365177 HE BB Heat Map View e Show Selected Cell Sample Setup a mio ml amp amp E Fit Eva Green at 20 C Ct x Layout Inlet based View HB Detector Setup a Detector Inlet Position a e gm 3 ps D Q i Tas Analyze Click the Analyze button To analyze a chip run file for the first time or After analysis parameter is changed Analysis Settings aPCR MCA E Peak Sensitivity 10 Y D Sil tabbed 2 Threshold Show No Call Log Graph Full Range
141. shokd ai af a nu LRL mihi M yey Chest a nul reb Reco oani ou fan arei the detesd C1 Geethaldla xF if n3 x7 ET om w wt 2 ga w uan om w w 42 25 i 95 w ET ga w w EE ga w uan ga w xf ET 0 21 w ET Dr w w 2 0 95 xf PeT 0 96 w xf EE 0 w EST om w al 0 5 3 Ind ET 85 rd al 4 25 og Id ET DT rd uf E E af EST wr a an iy a Pak 0 96 wf w 0 46 1 0 wt ns 1 a y hd on w s on ail Ld i 43 DE wf iEn DE w wt Ed m Ld iEn w Ld ms 1 La iEn GE w Ld om La iEn om ww Ld 1 Ld iS En GE w w x oa Cl iEn GE w u DE a A Pi E ad E Viewing Delta C Data in the Heat Map In addition to viewing A C and A A C data in the Results Table view A C data in the Heat Map 1 Click Analysis View 2 Click Analyze if necessary 3 Click Heat Map View from the Results Table menu Analysis Views Results Table El Show Results Table Image View Heat Map view 110 Fluidigm Real Time PCR Analysis Software User Guide Viewing Delta Ct Data in the Heat Map 4 Select FAM MGB Delta C Sample from the menu Reference cells Analysis Views GD Bil Heat Map view amp Show Selected Cell MGI A 9 Famme Delta cts v inlet based view E peg FAM MGB Ct Detector Inlet Position at E a 03 retre ihi Hh MM wes j FAM MGB J sjRjeje soportes Deta ct neselTETSERTEISETRTSRIETST amp e s e ECEEEGEEEEEE Lies CCE RRECCROCC ee LL 1 1 1056 6 1 ee
142. st Tr TIBUS RS 95 Paris Og ras Unkriren 1 Teal 17 Fe 095 Parii agaras Unkriren 1 Tiit ATTIS 095 Paws agaras Unkrarwn 1 Tes 17 84150677 053 Pais D r5 Unknawn 1 Test RE 034 Pass a ofr ras Unikraywn 1 Test LEAS oeraf 95 Pass B ode vas Unknown 1 Tet VS OS Paes OW Unkrnawn 1 Test 1T BiTi 053 Pass Dar ras Unknown i amp Teal 17 31054908 035 Pais aars Unkrarem 1 LL FEM ireen D 96 Parii garias Unkriren 1 Teal 17 IDES 092 Pari O D vas Unkrarwns 1 Tes 1BCET 1222 055 Pais ag ras Unknagwn M Test 18 S 13 Pass a ofr res Unkmown T Test EET cz 24 O91 Pass Oe vas Drinn 1 Test aa Paes EA Unine 1 Test 10 See DA Pass EA Ukren 1 Teal 1S 1560S 0 778556 Pais OO is Unkrusun 1 Tet 1314121213 0 7442983 Piit O Or is Lirias 1 Teal 19 ISS 0 SRE Fail agaras Unkrarens 1 Tes gn Fad a0 ras Fluidigm Real Time PCR Analysis Software User Guide 103 Viewing Chip Run Data in the Data Analysis Software The exported csv file below was saved from the Heat Map view Haatmap Rasuli bebet baibed view 5 Quality Thresheld OS t T PAA Ct B 1 2 x 4 5 E T B 8 1 i 12 13 13 1 1737 17 37 iF A43 17 47 17 45 i AT 17 55 175 1753 17 5 17 45 175 17 41 14 7 17356 17 58 AT AT 17 55 17 4 17 657 17 55 17 51 1753 17 48 1T 2 17 57 17 48 15 3 Tx Ta IT a T 17 89 17 29 1753 17 3 17 A 17 47 17 55 175 17 52 i a Ta T7 3X i To Weal i 17 43 Ta ma 17 45 lf af as T7 48 17 5 Tas 17 48 Li c T3 17 35 LR ws 17 44 L
143. t changes in the legend The examples below illustrate that the greater the number of color segments the finer distinction between legend values 1 Color Segment Color Lookup Editor Color Scheme Tm YellowT oBlue v Data Range Number of color segments Minimum Value TAN Maximum Value 35 00 Fluidigm Real Time PCR Analysis Software User Guide 81 Viewing Chip Run Data in the Data Analysis Software 10 Color Segments Color Lookup Editor Color Scheme Tm YellowT oBlue v Data Range Number of color segments Minimum Value Maximum Value Changing Minimum Values Change the minimum value when you want to exclude a segment from the lower range For example changing the value from 1 to 10 excludes any C value from 1 to 10 as the example below illustrates 1 Enter a value 2 Click OK Segments with values from 1 to 10 have been eliminated as the minimum acceptable value is now greater than 10 As a result the lowest value on the legend is now over 10 Color Lookup Editor Color Scheme Tm rellewiT oBlue Data Range Number of color segments Minimum Value Maximum Yalue Save Changing Maximum Values 82 Change the maximum value when you want to exclude a segment from the higher range from 1 to 39 For example changing the maximum value from 35 default value to 10 any C or T values above 10 are excluded gray area a
144. tection Reagents Supported Detection Reagents We support the following detection reagents with the BioMark HD System Probe Types e FAM MGB VIC MGB e FAM TAMRA e FAM non fluorescent quencher Additional Probe Types Fluidigm does not support other probe types at this time however additional probe types may be run with the BioMark HD System using the following guidelines Fluorophores With Excitation Wavelengths Emission Wavelengths between 465 and 505 nm And between 500 and 550 nm between 510 and 550 nm And between 540 and 600 nm PCR Master Mixes The protocol described in this manual uses TagMan Universal PCR Master Mix 2X Applied Biosystems PN 4304437 If you choose to use master mixes other than TaqMan Universal PCR Master Mix you may have to alter the protocol described in this manual Contact Fluidigm Technical Support for additional information IMPORTANT You must use a passive reference 1 Fluidigm recommends that you only use TagMan probes and or other licensed PCR assay reagents from authorized sources If you have any questions regarding whether you have a license to use particular reagents in PCR systems you should contact the appropriate licensor and obtain clarification and their permission if necessary For example certain probes and their use may be covered by one or more patents held by Applied Biosystems and or Roche Molecular Systems which may be contacted
145. ted Linear 72 Std3 SD9 Pn Std2 S04 ir the first time or PUR MT changed fi 4 Record 11 of 13 gt m lt 4 Record 1 a XR VicGUSEB 14 15 15 44 Slope y 1 95x 22 18 R2 0 5616 e of the Ct threshold 32 00 u ise a Manual methad n t threshold s 31 00 0 65 30 00 i 29 00 Linear v 2800 a User Data Global 2700 i E 6 0 03 26 00 l r 24 00 4 a 23 00 i 22 00 2100 UNDE Less e 0 0010 0 1000 0 0001 0 0100 1 0000 Concentration Calibrator 29 73 0 00 29 11 0 00 28 59 0 00 25 54 0 0039 26 04 0 0039 25 41 0 0039 23 78 0 0156 24 34 0 0156 24 59 0 0156 22 86 0 065 23 68 0 065 23 88 0 065 22 64 0 25 0 51 of 72 CJC Error 2 79 1 09 0 28 1 81 9 71 2 17 3 72 1 87 1 24 2 660 0 75 0 38 Laan aai Call KIKIKI KAII Corresponding plotted points changes to red the original call state c Go to the Analysis Views page d Click the Analyze button to re analyze the chip with these new parameters Fluidigm Real Time PCR Analysis Software User Guide KIS NOTE You can use the Call Redo or Undo buttons to revert back to 119 Viewing Chip Run Data in the Calibration Curve View e The resulting calibration curve is slightly modified NRC Weighted Linear s4 Std2 S04
146. the Active Window is Your Options Are Analysis Views Edit n e Ctri4 Z edc Ctrl OH Copy Ctrl C i Pass Ctrl G ag Fail Ctrl F ig No Call Ctrl I i5 Clear Ctri L ih Clear Al Ctrl A Results Table Edt Indo Ctrl Red tri 3 Copy Ctrl C a Pass Ctrl G a Fail Ctrl F io No Call Ctrl I i5 Clear Ctrl L i Clear All Ctrl 4 Image View dt Undc tri 2 Rede trl y Copy Ctrl C O Copy View Image p Save View Image Ww Pass Ctrl G amp Fail Ctrl F No Call Ctrl I i Clear Ctrl L 4 Clear all Ctri A Heat Map dt Undo Ctrl Z Red Ctrl Copy Ctrl C O Copy View Image p Save View Image WP Pass Ctrl G amp Fail Ctrl F iQ No Call Ctrl I 5 Clear Ctrl L 3 Clear All Ctri A View Select Chip Explorer and or Task to display these panes in your window Report Two reports are available Chip Preparation Report This report records the loading pattern for a chip run After creating a new chip run file use the Chip Preparation Report to record the data for hand pipetting 26 Fluidigm Real Time PCR Analysis Software User Guide Menus and Icons Fluidigm fis E Tia M A I Real Time PCR Analysis Chip Run Preparation Report p 7 siad cee Tm LINE T Lancia Oe el eee eet CLER LE sd LE ALS Ss ais LS T piei East S xam is ika rai Bi Jf we EEL Lanta LIN ea Erg e Install Test Report This report is only avail
147. the chip surface using scotch tape You are now ready for your chip run 150 Fluidigm Real Time PCR Analysis Software User Guide Exonuclease Exo Treatment Method MN Sample inlets Figure 2 Figure 2 96 96 Dynamic Array IFC sample and assay inlets Fluidigm Real Time PCR Analysis Software User Guide 151 Using the Data Collection Software 1 Double click the Data Collection Software icon on the desktop to launch the software 2 Click Start a New Run 3 Check the status bar to verify that the camera has a green light to indicate that it is ready 4 Remove the blue tape from the back of the chip if this was not done previously Place the chip into the reader Click Load Verify chip barcode and chip type Choose project settings if applicable Click Next Chip Run file a Select New o 0O N O0 Ul b Browse to a file location for data storage c Click Next 10 Application Reference Probes a Select Application Type Gene Expression b Select Passive Reference ROX c Select Probe Single probe d Select Probe type EvaGreen e Click Next 11 Click Browse to find the thermal cycling protocol files For BioMark HD e GE Fast 48x48 PCR4Melt v2 pcl e GE Fast 96x96 PCR4Melt v2 pcl For BioMark e GE 48x48 PCR4Melt v2 pcl e GE 96x96 PCR4Melt v2 pcl 12 Confirm Auto Exposure is selected 13 Click Next 14 Verify the chip run information 152 Fluidigm Real Time PCR Analysis Software User Guide Using
148. tion Levels of Unknown Samples Viewing Multiple Calibration Curves llle Fluidigm Real Time PCR Analysis Software User Guide 113 Viewing Chip Run Data in the Calibration Curve View Introduction The Calibration Curve View Module CCVM also known as standard curve isa view that allows the user to create calibration curves based on the C and known concentration differences of samples on the chip After calibration curves are created they are used to determine the approximate concentration of unknown Samples on the chip The approximate values are displayed in a table format For the CCVM to appear 1 Open an unanalyzed chip run See Opening an Existing Chip Run on page 29 for more information Select Analysis Views Click the Analyze button Select Sample Setup Click New to set up anew sample plate Choose SBS plate or Sample Inlet for your container type For more detail see Setting Up a Sample Plate on page 33 6 Select a Mapping option left or right side maps ulm W N Task 4 etup Click one of the following New to create a sample plate Export to save a plate for reuse Import to open an existing plate Plate Settings Source 96 Wellplate Name Barcode Mapping 4848SampeSBSSeLet Click this button to see mapping choices 7 Usethe Editor to annotate your sample cells Make sure at least two wells are Standard type essential
149. trument or software important to the successful outcome of your experiments ERR Q NOTE This convention highlights useful information L IMPORTANT This convention highlights situations or procedures that are Fluidigm Real Timel PCR Analysis Software User Guide 11 Related Documents This document is intended to be used in conjunction with these related documents e Fluidigm Data Collection Software User Guide PN 68000127 e Fluidigm IFC Controller Usage Quick Reference PN 68000126 Fluidigm 48 48 Fast Real Time PCR Workflow Quick Reference PN 100 2637 e Fluidigm 48 48 Real Time PCR Workflow Quick Reference PN 68000089 e Fluidigm 96 96 Fast Real Time PCR Workflow Quick Reference PN 100 2638 e Fluidigm 96 96 Real Time PCR Workflow Quick Reference PN 68000130 e Fluidigm Control Line Fluid Loading Procedure Quick Reference PN 68000132 e Fluidigm Gene Expression Specific Target Amplification Quick Reference PN 68000133 12 Fluidigm Real Timel PCR Analysis Software User Guide BioMark System Ree OP Luca ie he WK Rode dod RUE EERE IHG de OE Cao E d 14 eer er a e444 ee ee ee eae eS oe ee 14 Advantages of Real Time qPCR naaa 14 PLE FUGA MEN cle 4 43 he ee ror deed ode ER DED ERE EC X3 XR PR IE el 14 The Exponential Phase 1 0 cc ees 14 Advantages of Real Time qPCR TagMan Chemistry 00000 ee 13 BioMark HD System for Genetic Analysis leeren 16 High End Detection Optics wa
150. ws expand as shown below Chip Explorer 5 Chip Run Gene Expression A Analysis Views Sample Setup cs Sample Mapping View B Detector Setup Analysis Views E x Results Table gal Show Selected Rows ce Type gees ID E Full Collapse Experiment Information Se Clear Grouping Chamber Sample FAM MGB ID Name Type rConc Name Type Name 532 Name 533 Name 534 Name 535 Analysis Views Chip Explorer ij Chip Run Gene Expression A Analysis Views eur Sample Setup amp y Sample Mapping View B Detector Setup Analysis Views D ix Results Table v d Show Selected Rows D 2 Results Table d Show Selected Rows Name ae Type LL Experiment Information Chamber Sample FAM MGB ID Name Type rConc Name Type a Name 501 2 Type Unknown E ID R48 CO1 R48 S01 Unknown 1 00 D01 Unknown E ID R48 C02 v R48 501 Unknown 1 00 D02 Unknown Name t Type aos ID Experiment Information Chamber Sample ID Name R48 501 i ID R48 C47 R48 501 E Name 502 Type Unknown i ID R47 CO1 R47 502 2 ID R47 CO02 R47 502 E ID R47 CO3 FAM MGB FAM MGB ct Type rConc Name Type Value Quality Call Unknown 1 00 D46 Unknown 17 21 0 90 wv Unknown 1 00 D47 Unknown 17 26 0 98 v Unknown 1 00 DO1 Unknown 18 71 0 88 v a v Unknown 1 00 DOZ Unknown 18 24 0 86 v 3 Collapse

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