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TaqMan® Human Cytokine Card

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1. Step Action 1 Select from the following If setting the threshold for Then the first card in an follow steps 2 5 of this procedure experimental series all subsequent cards a Click the Use Threshold text field of the Threshold box ao Lj Click here and ft Souter SESE SE type the value Bae 2 0 1 for the threshold Start z Stop 15 b Type the threshold value for the current dye layer determined in step 4 of this procedure from the first card run in the series c Click Update Calculations The SDS software updates the C and standard deviation values 2 Identify the components of the amplification curve as shown in Threshold Value Basics on page 4 5 Data Analysis 4 7 4 8 Data Analysis To set the threshold value for the VIC dye layer continued Step Action 3 Click and drag the threshold line so that it is Above the background noise Below the plateaued region Within the exponential phase of the amplification curve 10 2 1o 3 a Cycle Number 35 4 Record the threshold value Threshold Cycle Calculation Apply the same threshold value to the VIC dye layer of all subsequent cards in the comparison Below the plateaued region Within this range E Above the background The software displays the threshold value in the Use Threshold text field within the Threshold
2. Load each TaqMan Human Cytokine Card with sample specific PCR reaction mix made with a single cDNA sample Cytokine cards are not designed to evaluate multiple cDNA samples simultaneously Run each TaqMan Human Cytokine Card within 30 minutes of loading it When loaded cards sit the TaqMan probes and primers within the wells of the card begin to diffuse into the adjoining channels The diffusion of critical reagents diminishes the potential signal that can be generated during PCR and can therefore affect the results of the experiment Run all TaqMan Human Cytokine Cards for a single experiment on the same ABI PRISM 7700 instrument Running all cards on the same instrument ensures a high degree of reproducibility and consistency in the resulting data Load 300 uL of sample specific PCR reaction mix per card to ensure adequate filling Smaller volumes may result in insufficiently filled cards Do not attempt to refill partially filled cards Upon contact the sample specific PCR reaction mix resuspends the dried TaqMan probes and primers within the wells of the card When the partially filled card is evacuated these reagents are carried away with the solution Recommended Quantity Preparing a Sample Specific PCR Reaction Mix The following table lists the recommended range of human total RNA converted to cDNA for the PCR Sample Sample Quantity per Card 300 yL sample specific reaction mix
3. 0 4 12 Eliminating Wells from the Analysis 0 4 13 Exporting the Analyzed Run as a Results File 0 4 14 Exporting a Results File 2 0 0 0 eee eee eee 4 14 vi 5 Interpreting Results OVEDVICW bocce baat see Ae age ate Cae REE eee ed 5 1 Where You Are in the Procedure 00 00 0005 5 1 Calculating Relative Cytokine Gene Expression 5 2 Rationale nisione cet 5 oR TG Gy See E le Rec REE N 5 2 Reference sits 2 hasdini telow Stud ens elas ead Ma leek AN le alee 5 2 Configuring the ABI PRISM 7700 Relative Quantification Software 5 3 Creating a Template for Card Analyses 5 3 Interpreting Relative Quantification Results 004 5 4 About the Results Window 0 2 0 0 eee eee eee eee 5 4 About the Cytokine Gene Expression Profile 5 5 KAARIS jee iusaeh Canad vit Alain ne GR a Rite se ae A 5 5 YARIS hese ed sae aod aie a a sd Gage eye oe 5 5 Sample Bars aeiicke nests coast bls Wee eudes Gand eel aps Rene 5 6 Troubleshooting QVERVIOW Sino 08 Seren Bio nid ail ayn Bas Beale eee eR eee 6 1 Loading the Card issa iee ad ae ee sate aes Soda bee eens 6 2 Data Analy sisii 2 isc odes fede ta a Baers bean eee es 6 5 Interpreting Results 2 3 2 ces seek ies See gan be SS He es 6 10 Theory of Operation OVERVICW srp whee a eee are eae Beet iSe ale ete Goes A 1 TaqMan Human Cytokine
4. Troubleshooting Overview This chapter provides information on how to troubleshoot the following areas Loading the card Data analysis Interpreting the results Troubleshooting 6 1 Loading the Card Troubleshooting Card Loading Observation Vacuum is not reaching the proper level lt 600 Microns The sample does not enter the card when the actuator is pulled The sample partially enters the card when the plunger is pulled Large bubbles appear in the card immediately after filling Note Cards typically contain small bubbles after thermal cycling Possible Cause s The ABI PRism Card Filling Station is not completely closed The tubing between the pump and the Filling Station contains a loose connection The battery in the vacuum gauge is low The vacuum pump requires maintenance The ABI PRisM Human Cytokine Card is not aligned properly inside the Filling Station The fill consumable and the reaction card are not aligned The card and fill consumable are not aligned properly inside the Filling Station Recommended Action Conduct the following a Press down on the Filling Station lid to ensure that it is fully closed b Check the tubing and connections for leaks c Test and change the battery in the vacuum gauge if necessary d If the problem persists verify that the cytokine card is positioned correctly within the Filling Station as
5. Total RNA converted to cDNA 60 ng 2 ug a 300 pL sample specific PCR reaction mix 150 uL cDNA sample 20X 18S Primer and Probe Mix H20 150 uL TaqMan Universal PCR Master Mix IMPORTANT Load only one sample specific PCR reaction mix CDNA sample 20X 18S Primer and Probe Mix TaqMan Universal PCR Master Mix per TaqMan Human Cytokine Card Applied Biosystems recommends running your calibrator sample first Note This procedure is optimized for TaqMan PCR Universal Master Mix To prepare a sample specific PCR reaction mix Step Action 1 2 Label a 1 5 mL microcentrifuge tube Retrieve a cDNA sample from the freezer see step 3 on page 2 6 If frozen thaw the sample by rolling it between your fingers Transfer the recommended quantity of cDNA sample up to 150 pL endogenous control to the labeled microcentrifuge tube IMPORTANT To avoid cross contamination of the reverse transcription products slowly and carefully remove the cap from the thermal cycling tube Dilute the sample to 120 uL with RNase free deionized water Add 150 uL of TaqMan PCR Universal Master Mix 2X to the microcentrifuge tube containing the dilute cDNA sample CHEMICAL HAZARD TaqMan Universal PCR Master Mix may cause eye and skin irritation It may cause discomfort if swallowed or inhaled Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves
6. m Plateau phase e eiit Exponential i f sSSES5 phase s Threshold EEEE Sela Donan m Background Eee 4 _ Cycle Number Guidelines for setting the threshold value are explained on the following page Data Analysis 4 5 Guidelines for To set the threshold correctly Setting Thresholds Set the threshold value within the exponential phase of the 4 6 Data Analysis semi logarithmic scale amplification plot The exponential phase occurs within the range of data points that increase linearly when graphed on a semi log plot Set identical FAM thresholds for all cards in the same comparison The ABI PRISM 7700 Relative Quantification Software cannot analyze data from card runs that have different FAM threshold values Set identical VIC thresholds for all cards in the same comparison The ABI PRISM 7700 Relative Quantification Software cannot analyze data from card runs that have different VIC threshold values Setting the Threshold for the VIC Dye Layer IMPORTANT The baseline values for TaqMan Human Cytokine Card runs cannot be set manually Version 1 7 1 and later of the SDS software automatically calculates and sets the baseline for card runs To set the threshold value for the VIC dye layer
7. Add 30 uL of 20X 18S Primer and Probe Mix Cap the microcentrifuge tube and mix the solution by gentle inversion Centrifuge the tube to eliminate air bubbles from the mixture PCR 3 5 To prepare a sample specific PCR reaction mix continued Step Action 9 Place the sample specific PCR reaction mix on ice and prepare a TaqMan Human Cytokine Card as described on page 3 9 3 6 PCR Loading the TaqMan Human Cytokine Card About The following figure shows an exploded view of an ABI PRiSM card ABI PRISM Cards Number Component Description 1 Reaction card Acts as the vessel for the PCR The consumable consists of a series of 96 interconnected wells pre loaded with dried TaqMan probes and primers 2 Adhesive flap Used to seal the reaction card after it has 3 Adhesive backing been filled with sample specific PCR reaction mix 4 Fill reservoir A reservoir for the sample specific PCR reaction mix before it enters the card 5 Fill consumable A disposable component that channels fluid from the fill reservoir into the reaction card 6 Alignment holes Aid in aligning the card within the ABI PRISM Filling Station PCR 3 7 Guidelines for Follow the guidelines below to ensure proper loading of the card Loading Cards Allow a card to adjust to room temperature before loading it 3 8 PCR Low temperatures will prevent efficient flu
8. Qoo o Repeat this procedure for each cDNA sample Data Analysis Analysis of raw data from the card run Note This step is performed using the ABI PRISM 7700 Sequence Detection Systems Software a Set the threshold values for the VIC and FAM dye layers b Eliminate outlying amplification Optional c Export the analyzed data as a results file Repeat the analysis for each card run Interpreting Results Analysis of data from multiple results files Note This step is performed using the ABI PRISM 7700 Relative Quantification Software a Import results from the card runs b Eliminate outlying amplification c Analyze the results Introduction 1 3 Virtual Dye Layers Genomic DNA Contamination 1 4 Introduction The TaqMan Human Cytokine Card permits the amplification of cytokine and endogenous control cDNA using a multiplexed fluorogenic 5 nuclease assay The assay consists of two reactions each a complete PCR system with corresponding probe and primers The fluorogenic probes of the multiplexed assay function as follows Probes labeled with the FAM dye detect the amplification of 24 cytokine cDNA targets Probes labeled with the VIC dye detect the amplification of cDNA generated from the 18S rRNA endogenous control The following conceptual figure illustrates the virtual dye layer configuration of a TaqMan Human Cytokine Card GR1569 TaqMan Cy
9. To activate spectral compensation Step Action 1 Select Advanced Options from the Diagnostics submenu off of the Instrument menu The Advanced Options dialog box opens 2 Click the Use Spectral Compensation for Real Time check box from the Miscellaneous box Set 7700 Exposure Time for Plates for Cards Use Spectral Compensation for Real Time Miscellaneous Click here Use Spectral Compensation for Endpoint Reference ROX 3 Click OK Non Fluorescent To setup for a non fluorescent quencher at the 3 end of the probe Quencher Step Action 1 From the Sample Type pop up menu select Sample Type Setup The Sample Type Setup dialog box appears Acronym Name Sample Type Setup Color Reporter Pe _ Internal Positive i z IPC internal Positive CL a2 _ Allele 2 xtc No Template Control B ci nac No Amplification unen Unknown E Reference E o Quencher a rara TS ok 2 Complete the probes b Click OK a Uncheck the box next to Quencher when using TagMan MGB The dialog box closes and the plate read window becomes active Sample Type Setup dialog box Setting the Baseline Values Automatic The baseline values for TaqMan Human Cytokine Card runs cannot be Baseline set manually Version 1 7 1 and later of the ABI PRISM 7700
10. 4 B Well FAM Ct 35 2 35 4 3 35 11 Click cell B1 and type Ct FAM 12 13 Repeat steps 3 7 to copy the VIC C values in cells F111 F207 from the results file and paste them into cell C1 of the new Excel file FAM Ct 1 2 35 12 96 3 Click cell C1 and type Ct VIC Calculating AC AC is calculated with the equation AC Cy fam Crvic TO calculate AC s the formula must be entered into column D To enter the equation into the spreadsheet Step Action 1 Click cell D2 2 Type the equation B2 C2 and press Return 3 Click cell D2 4 From the Edit menu select Copy 5 Select cells D3 D97 by clicking and dragging the mouse cursor down the spreadsheet document 6 From the Edit menu select Paste Excel pastes copies of the equation into cells D2 D97 of the new spreadsheet The program automatically calculates AC values E ee a ee ae ee ee ee a well FAM Ct IC Ct ACT Ea 12 17 22 83 ES 2 35 12 98 22 02 4 3 35 13 1 21 9 7 Click cell D1 and type AC 8 From the File menu select Save As Save the spreadsheet document B 6 Demonstrating Performance with Control RNA Creating an Now that AC values have been calculated from the cytokine card data Average AC Table the results must be organized under the cytokines they detect To create a Median AC table Step Action 1 Click cell F1
11. To Reach Technical Support by Phone or Fax Outside N America C 4 To Reach Technical Support Via the Applied Biosystems Web Site C 6 To Obtain Technical Documents 0 00 00 000000 C 6 To Obtain Customer Training Information C 7 D References E Limited Warranty Statement Introduction Getting Started Quickly Product Overview If familiar with the theory behind TagqMan Human Cytokine Card chemistry or ABI PRISM 7700 Sequence Detection System data collection read the sections of this protocol listed below They contain the minimum amount of reading required to conduct TaqMan Human Cytokine Card experiments Chapter 1 Introduction Designing TaqMan Human Cytokine Card Experiments on pages 1 7 to 1 8 Preventing Contamination on page 1 9 Materials and Equipment on pages 1 10 to 1 11 Chapter 2 Reverse Transcription Chapter 3 PCR Chapter 4 Data Analysis Chapter 5 Interpreting Results If unfamiliar with the concepts behind the cytokine card chemistry or ABI PRISM 7700 data collection read the following sections in addition to the ones listed above Chapter 1 Introduction on pages 1 1 to 1 6 Appendix A Theory of Operation The TaqMan Human Cytokine Card is a research tool for profiling human cytokine gene expression using the Comparative C Method of relative quantification The card evaluates a single cDNA sample genera
12. IL 10 IL 10 IL 10 L 1ap36 Has L 1240 L 1240 L 12p40 L 12p40 Cabrator Cabrator Cabrator Calbrator era Ciar Career cake ator Calibrator Calibrator Calbrator Calbrator IL 18 IL 13 IL 18 IL 13 IL 15 IL 15 IL 15 IL 15 IL 17 IL 17 IL 17 IL 17 Calibrator Cabrator Calbrator Calbrator Calbrator Calbrator Calibrator Cabrator Calibrator Cabrator Calibrator Calbrator IL 18 IL 18 IL 18 IL 18 G CSF G CSF G CSF G CSF GM CSF GM CSF GM CSF GM CSF Cabrator Cabrator Calbrator Calbrator Calibrator Cabrator Calbrator Cabrator Calbrator Calbrator Cabrator Cabrato M CSF M CSF M CSF M CSF FMg maf FN g mal Fig mal Fg maj LT beta LT beta LT beta LT beta Cabrator Cabrator Cabrator Calbrator Cabrator Cabrator Calibrator Cabrator Cabrator Cabrator Calbrator Calibrator TGF beta TGF beta TGF beta TGF beta TNF alpha TNF alpha TNF alpha TNF alpha TNF beta TNF beta TNF beta TNF beta ator Calbrator Calbrator Calbrator Cabrator Calibrator Calbrator Calbrator Calbrator Calbrator Calbrator Calbrator Interpreting Results 5 3 Interpreting Relative Quantification Results About the The ABI PRISM 7700 Relative Quantification Software analyzes the data Results Window from the exported results files and displays the analysis in the Results window The figure below illustrates a typical results window generated
13. from the Analysis menu 2 Select VIC from the Reporter pop up menu Viewer All IC Reporter The VIC Endogenous Control Assay Amplification Plot appears 4 4 Data Analysis AR Threshold Value Before exporting the data for relative quantification the threshold value Basics of each TaqMan Human Cytokine Card run must be adjusted for 10 10 10 102 103 quantification For any analysis of TaqMan Human Cytokine Cards accurate quantification depends on the uniformity of the threshold values If the threshold values are not adjusted properly or if they differ between cards in a comparative experiment the resulting data will be invalid for relative quantification The threshold value must be determined once for each relative quantification experiment typically with the results from the first card run Subsequent runs within the same experiment must use the value determined from the calibrator card Occasionally the threshold value for an analysis group must be readjusted to compensate for a later run In those cases the readjustment is made to the outlying card and then applied to the others The threshold value of the calibrator card run can be determined by viewing the amplification plots for the card using the SDS software The figure below shows a typical semi logarithmic VIC amplification plot with the correct threshold setting Amplification VIC Example Product amplification
14. Perform reverse transcription as described in Chapter 2 using 2 ug of TaqMan Human Control Total RNA in a 100 pL reaction 2 Make a sample specific PCR reaction mix using 150 pL TaqMan Universal PCR Master Mix 30 uL of 20X 18S Primer and Probe Mix 118 uL water and 2 pL cDNA as described in Chapter 3 PCR Then fill and run a TaqMan Human Cytokine Card with the reaction mix B 2 Demonstrating Performance with Control RNA To verify the C values continued Step Action 3 After the run is complete activate the Spectral Compensation feature a Select Advanced Options from the Diagnostics submenu off of the Instrument menu The Advanced Options dialog box appears b From the Miscellaneous group box click the Use Spectral Compensation for Real Time check box c Click OK Select Analyze L from the Analysis menu The SDS software analyzes the raw data and displays an amplification plot log AR vs Cycle From the Reporter menu select VIC The SDS software displays the amplification plot log AR vs Cycle for the VIC dye layer Threshold Cycle C From the Viewer menu select Cy vs Well The SDS software displays the Threshold Value C vs Well Position view for the VIC dye Because of the high abundance of rRNA performance integrity across a entire card can be best assessed by examining the Cy from the 18S rRNA endogenous control The card
15. S only or 1 781 271 0045 1 781 275 8581 LC MS Applied Biosystems MDS Sciex 1 800 952 4716 1 508 383 7899 a 5 30 AM to 5 00 PM Pacific time b 8 00 AM to 6 00 PM Eastern time c 9 00 AM to 5 00 PM Eastern time To contact Applied Biosystems Technical Support or Field Service outside North America use the telephone or fax numbers below Region Telephone Fax Eastern Asi a China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 or 86 1064106617 86 800 8100497 Hong Kong 852 2756 6928 852 2756 6968 India New Delhi 91 11 653 91 11 653 3138 3743 3744 Korea Seoul 82 2 593 6470 6471 822593 6472 Malaysia Petaling Jaya 60 3 79588268 60 3 79549043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe C 4 Contacting Technical Support Region Telephone Fax Austria Wien 43 0 1 867 35 750 43 0 1 867 35 75 11 Belgium 32 0 2 532 4484 32 0 2 582 1886 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 1010 49 0 6150 101 101 Italy Milano 39 0 39 83891 3
16. and type Target 2 Click and type the following entries into the designated cells Click Cell Type Click Cell Type F2 IL 1alpha F14 IL 13 F3 IL 1beta F15 IL 15 F4 IL 2 F16 IL 17 F5 IL 3 F17 IL 18 F6 IL 4 F18 G CSF F7 IL 5 F19 GM CSF F8 IL 6 F20 M CSF F9 IL 7 F21 IFN gamma F10 IL 8 F22 LT beta F11 IL 10 F23 TGF beta F12 IL 12p35 F24 TNF alpha F13 IL 12p40 F25 TNF beta Demonstrating Performance with Control RNA B 7 To create a Median AC table continued Step Action 3 Click and type the following equations into the designated cells Cell Type Cell Type G2 MEDIAN D2 D5 G14 MEDIAN D50 D53 G3 MEDIAN D6 D9 G15 MEDIAN D54 D57 G4 MEDIAN D10 D13 G16 MEDIAN D58 D61 G5 MEDIAN D14 D17 G17 MEDIAN D62 D65 G6 MEDIAN D18 D21 G18 MEDIAN D66 D69 G7 MEDIAN D22 D25 G19 MEDIAN D70 D73 G9 MEDIAN D30 D33 G21 MEDIAN D78 D81 G10 MEDIAN D34 D37 G22 MEDIAN D82 D85 G11 MEDIAN D38 D41 G23 MEDIAN D86 D89 G12 MEDIAN D42 D45 G24 MEDIAN D90 D93 G8 MEDIAN D26 D29 G20 MEDIAN D74 D77 G13 MEDIAN D46 D49 G25 MEDIAN D94 D97 Click cell G1 and type MedianACt Compare the AC s of the targets in the spreadsheet to the values in the table below The card is perfo
17. connection between the reaction card and the fill consumable Step Action 1 Turn off the vacuum pump 2 Using a pipet temporarily remove the sample from the reservoir and store it on ice Open the Filling Station Remove the TaqMan Human Cytokine Card from the Filling Station Inspect the connection between the reaction card and the fill consumable The consumable design includes two alignment pins and a small aperture through which the sample enters the card When situated correctly the pins and aperture align with three holes on the keyed end of the reaction card The following figures illustrate this card consumable relationship from opposing angles If the card components do not align as shown above readjust the position of the fill consumable to restore the connection Load the TaqMan Human Cytokine Card into the ABI PRISM Filling Station and re attempt the loading procedure Data Analysis Troubleshooting Data Analysis Observation Possible Cause Recommended Action Inthe Amplification Plot view the fluorescent signals show little or no growth Figure 6 1 AND In the Multicomponent View all well signals slowly degrade to background by the end of the run Figure 6 2 The heated cover was not tightened adequately Remember to tighten the heated cover completely for future runs The O ring on the lens plate that seals the card fill p
18. explained on page 6 3 e If the problem persists verify the integrity of the connection between the reaction card and fill consumable as explained on page 6 4 Verify that the cytokine card is positioned correctly within the Filling Station as explained on page 6 3 6 2 Troubleshooting Verifying the Position of the Card Within the Filling Station To verify that the card is positioned correctly within the Filling Station Step Action 1 Turn off the vacuum pump 2 Using a pipet remove the sample from the reservoir and store it temporarily on ice 3 Open the ABI PRISM Filling Station 4 Inspect the card for the following The adhesive flap must be folded away from the fill consumable The holes in the card s fill consumable must align with the pegs in the bay of the ABI PRISM Filling Station 5 Reposition the cytokine card and re attempt to load the card IMPORTANT Do not attempt to refill partially filled cards Upon contact the sample specific PCR reaction mix resuspends the dried TaqMan probes and primers within the wells of the card When the partially filled card is evacuated these reagents are carried away with the solution Troubleshooting 6 3 6 4 Troubleshooting Verifying the Consumable Card Connection The connection between the reaction card and the fill consumable is crucial for loading TaqMan Human Cytokine Cards To verify the integrity of the
19. from TaqMan Human Cytokine Card data Note The example profiles Shown below and on the following page were created using run data from T cell total RNA samples kindly provided by Dr R de Waal Malefyt DNAX Research Institute Palo Alto California USA Pee Quant File Cytokine card Results F Cytokine Gene Expression Profile M Results table Element Description Gene Expression A graphic representation of the results of the relative Profile quantification calculations displayed on a semi logarithmic scale The elements of the profile are described on the following page Results Table A numerical representation of the sample data at various stages of the relative quantification calculation Note See the ABI PRISM 7700 Relative Quantification Software User s Manual for information on the calculation 5 4 Interpreting Results About the The ABI PRISM 7700 Relative Quantification Software displays the Cytokine Gene results of the cytokine assays as the normalized mRNA level in test Expression Profile samples relative to the normalized level of that mRNA in the corresponding calibrator sample The gene expression profile is a graphic representation of the results of the relative quantification calculations The gene expression profile is located in the upper panel of the Resu
20. graph below the threshold cycle occurs when the Sequence Detection Systems software begins to detect the increase in signal associated with an exponential growth of PCR product Rnt Sample Rn Threshold Baseline GRO757 T T T T o 5 10 15 20 25 30 35 Cycle Number Al e A 6 Theory of Operation Demonstrating Performance with Control RNA Overview TaqMan Human Control Total RNA is available from Applied Biosystems for demonstrating the performance of the TaqMan Human Cytokine Card This appendix includes the performance guarantee statement and protocols for analyzing the Control RNA data Demonstrating Performance with Control RNA B 1 System Performance Guarantee Statement If TaqMan Human Control Total RNA is run in the card using the conditions in the following protocol the average C value for the 18S endogenous control will be fewer than 12 cycles and the AC values for five cytokine targets will be as follows Target ACy IL 10 Below 20 Lymphotoxin B Below 20 TGF B Below 17 TNF a Below 20 TNF B Below 17 Protocol for Analyzing TaqMan Human Control Total RNA Verifying Cy Card performance can be verified immediately after running a control Values Using the card by examining the threshold cycle C values of each reporter dye SDS Software layer within the Sequence Detection System SDS software To verify the C values Step Action 1
21. the ABI PRISM 7700 Instrument continued Step Action 4 Place the lens plate on top of the card so that the holes in the plate fit over the alignment pins on the sample block IMPORTANT Make sure that the lens plate is oriented so that the face with the This Side Up label is visible when the plate is in place Alignment pin 5 Slide the cover over the sample block and tighten the lid Note Because the dimensions of the card sandwich differ from those of a standard plate the white alignment line on the tightening knob may not turn to the same position as with a standard plate when tight IMPORTANT Tighten the cover as much as possible to ensure that the card is adequately sealed and uniformly heated Running TaqMan Human Cytokine Cards About SDS Plate Documents Using a Template Every TaqMan Human Cytokine Card run on the ABI PRISM 7700 Sequence Detection System requires the creation of a card specific plate document within the instrument software The 7700 instrument uses the plate document to organize and store the fluorescence data gathered during the PCR Plate documents contain the following information Dye layer setup Target sample configurations Thermal cycling parameters Data collection parameters A template file alleviates the need for repetitious construction of TaqMan Human Cytokine Card plate documents Template files are identical to plate document files except that they do n
22. 235 cycles A test sample produces an Average C lt 35 cycles At this level the calibrator sample does not contain enough cytokine target cDNA for an accurate comparison Therefore the stated expression level is the minimum possible value for the associated test sample Note The software displays gt in the cells of the results table that correspond to the values displayed in the bar graph Maximum target gene expression level Displayed when A test sample produces an Average C 235 cycles A calibrator sample produces an Average C lt 35 cycles At this level the test sample does not contain enough cytokine target cDNA for an accurate comparison with the calibrator sample Therefore the stated expression level is the maximum possible value for the sample Note The software displays lt in the cells of the results table that correspond to the values displayed in the bar graph Expression levels cannot be determined from the given data Displayed when both of the following conditions are met A calibrator sample produces an Average C 235 cycles A test sample produces an Average C 235 cycles At these levels the calibrator and test samples do not contain enough cytokine target cDNA for an accurate comparison Because both signals are insufficient the software cannot accurately evaluate the expression levels Note The software displays Insufficient Signal in the corresponding cells of the results table
23. 4404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 ABI PRISM and its Design Applied Biosystems and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries ABI is a trademark of Applera Corporation or its subsidiaries in the U S and certain other countries AmpErase AmpliTaq AmpliTaq Gold GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems Inc Macintosh is a registered trademark of Apple Computer Inc All other trademarks are the sole property of their respective owners 07 2001 4307577 Rev C Contents 1 Introduction Getting Started Quickly 0 cee eee eee 1 1 Product Overview sis s nanao keni age Stas eh eee geet 1 1 TaqMan Human Cytokine Card Design 0 0 1 2 Procedure Flowchart 0 0 0 cece eee ee eee eee 1 3 Virtual Dye Layers s cis ncaiete nse Sasi pace Ged pede pads Gat 1 4 Genomic DNA Contamination 00 020 1 4 Competition Between Multiplexed Reactions 1 5 Quality Control ssaa eh asatisd sa ee Wes ae eds 1 5 System Performance Guarantee 0 000002 eae 1 6 Designing TaqMan Human Cytokine Card Experiments 1 7 About the Comparative Cr Method of Relative Quantification 1 7 Significance of the Calibrator Sample 1 7 Design Guidelines niss woos as reaa oie hi sade Wa eee o
24. 54 Argentina 800 666 0096 55 11 5070 9694 95 Chile 1230 020 9102 55 11 5070 9694 95 Uruguay 0004 055 654 55 11 5070 9694 95 To contact Technical Support through the Applied Biosystems web site Step Action Go to http www appliedbiosystems com Click SERVICES amp SUPPORT at the top of the page then click Frequently Asked Questions Click Contact Support in the contents list at the left of the screen Click your geographic region for the product area of interest In the Personal Assistance form enter the requested information and your question then click Ask Us RIGHT NOW In the Customer Information form enter the requested information then click Ask Us RIGHT NOW Within 24 to 48 hours you will receive an e mail reply to your question from an Applied Biosystems technical expert To Obtain You can obtain technical documents such as Applied Biosystems user Technical documents MSDSs certificates of analysis and other related Documents docume nts for free 24 hours a day You can obtain documents By telephone Through the Applied Biosystems web site C 6 Contacting Technical Support Ordering Documents by Telephone To order documents by telephone 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note The
25. 9 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 12 06 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 392400 31 0 180 392409 or 31 0 180 392499 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 European Managed Territories EMT Africa English speaking Johannesburg South Africa 27 11 478 0411 27 11 478 0349 Africa French speaking Paris France 33 1 69 59 85 11 33 1 69 59 85 00 India New Delhi 91 11 653 3743 91 11 653 3744 91 11 653 3138 Poland Lithuania Latvia and 48 22 866 40 10 48 22 866 40 20 Estonia Warszawa For all other EMT countries not 44 1925 282481 44 1925 282509 listed Central and southeast Europe CIS Middle East and West Asia Japan Contacting Technical Support C 5 To Reach Technical Support Via the Applied Biosystems Web Site Region Telephone Fax Japan Hacchobori Chuo ku 81 3 5566 6230 81 3 5566 6507 Tokyo Latin America Caribbean countries Mexico and 52 55 35 3610 52 55 66 2308 Central America Brazil 0 800 704 9004 or 55 11 5070 9694 95 55 11 5070 96
26. BUFFER Buffer Blank Sample Type Setup Not In Use 3 Repeat steps 1 2 for the VIC dye layer outlying wells Data Analysis 4 13 Exporting the Analyzed Run as a Results File Exporting a To analyze data from the TaqMan Human Cytokine Card you must Results File export the results of the card run to a results file The SDS software can export results data from a sequence detection run in a tab delimited format that is compatible with the ABI PRISM 7700 Relative Quantification Software To export the run data to an SDS results file Step Action 1 Select Results 3 H from the Export submenu off of the File menu Raw Spectra Pure Spectra Page Setup Multicomponent Print RP Clipped Extension Phase 3E it S Results H Experimental Report 2 Click the Export result data as text box and type a name for the exported file Export result data as ftataresuts 3 Click the Export All Wells radio button Export All Wells Q Export Selected Wells 4 Click Export The SDS software exports the data The 7700 data file icon is shown below data results 5 Close the SDS software 4 14 Data Analysis Interpreting Results Overview The ABI PRISM 7700 Relative Quantification Software calculates relative cy
27. Card Chemistry 005 A 2 RIEPCR pose isd Meals tnaa Ioan eh eae ek a A 2 Basics of the 5 Nuclease Assay 0 c cece eee eee ee A 2 About AmpliTaq Gold DNA Polymerase A 4 Multicomponent Analysis 0 0 0 eee eee ee eee A 4 Fluorescent Detection 0 2c eee eee A 4 Passive Referente sac on Sivas ees see bad bes abd a A 4 Normalization soare saeia ta e gael ee ia ee ae ee A 5 R al Time Detection 2 2 Ges h0ei veyed ee als Sele tues care ee A 6 B Demonstrating Performance with Control RNA OVERVICW o einsi nia sila G ater N A eee E a e Hiss atlanta eaten ah B 1 System Performance Guarantee 0 0 cece eee eens B 2 Stateme t ess dae alone pclae etre e aa dress ed ere ene ys B 2 Protocol for Analyzing TaqMan Human Control Total RNA B 2 Verifying Cy Values Using the SDS Software B 2 Verifying the Expression Profile of Control RNA B 5 Transferring Data from the Results File B 5 Calculating AC Pisin cece acer ate ea ee eee aha pe eee eee B 6 Creating an Average ACy Table 00 00 0000 8 B 7 C Contacting Technical Support Technical Support i 00 5 an eee ee ee ee eng Mee ee gee SAE Eee C 1 Contacting Technical Support 0 0 00 02 0002 C 1 To Reach Technical Support by E Mail 0 C 1 To Reach Technical Support by Phone or Fax N America C 2
28. S Import Lab iew Format Raw Data 31 Page Setup Print 5 Using the browser navigate to the SDS Runs folder of the SDS software folder on the hard disk of your 7700 instrument 6 Select the Labview file from the problem run The SDS software labels Labview files by the time and date of the run Select the file with the correct creation date and time 7 Click OK The SDS software imports the data from the Labview file 8 Reanalyze the file as described in Chapter 4 Data Analysis Troubleshooting 6 9 Interpreting Results Troubleshooting Interpreting Results Observation Possible Cause Recommended Action Unable to import card file into the ABI PRISM 7700 Relative Quantification Software Files may not be the proper format Verify that the file you are attempting to import is a tab delimited results file exported from a card run and not the SDS run file If necessary export the files as explained in Exporting the Analyzed Run as a Results File on page 4 14 R SDS run file Files may not be configured with correct sample types TARG and ENDO While viewing the card run within the SDS software verify the following The TARG sample type must be applied to all wells in the FAM dye layer see page 3 19 for the FAM dye layer setup The ENDO sample type must be applied to all wells in the VIC dye layer see page 3 19 for the VIC dye layer setup If the ENDO and TARG sampl
29. Sequence Calculation Detection System SDS software automatically calculates and sets baselines for card runs IMPORTANT You must still set the baseline manually for plates run on the 7700 instrument The automatic baseline calling feature of SDS software version 1 7 1 is exclusive to card runs Data Analysis 4 3 Setting the Threshold Values Threshold Because TaqMan Human Cytokine Cards are used for comparative Requirements for analysis all cards in a comparison must Relative Share identical threshold settings for the VIC dye layer Suaniticavon Share identical threshold settings for the FAM dye layer The relative quantification calculation relies on a comparison of threshold cycle C values from separate ABI PRISM 7700 Sequence Detection System runs Because the threshold value affects the C s calculated by the SDS software the setting within each individual dye layer must be identical for all files in the comparison Note The ABI PRism 7700 Relative Quantification Software will not import multiple SDS results files that have different threshold settings Displaying To display the results on an amplification plot Results on an Amplification Plot Step Action 1 Select Analyze L from the Analysis menu The SDS software analyzes the raw data and displays the results in an amplification plot log AR vs Cycle If the software does not display the Amplification Plot select Amplification Plot 3 G
30. Systems 1 800 831 6844 press 3 then press 2a 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCRa 2 for TaqMan applications and Sequence Detection Systems including ABI Prism 7700 7900 and 57004 6 for the 6700 Automated Sample Prep Systema or 1 800 831 6844 then press 5a 1 240 453 4613 Voyager MALDI TOF Biospectrometry Workstations Mariner ESI TOF Mass Spectrometry Workstations MassGenotyping Solution 1 MGS1 System 1 800 899 5858 press 1 then press 3 1 508 383 7855 Biochromatography BioCAD SPRINT VISION and INTEGRAI Workstations and POROS Perfusion Chromatography Products 1 800 899 5858 press 1 then press 4 1 508 383 7855 Expedite Nucleic Acid Synthesis Systems 1 800 899 5858 press 1 then press 5 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 press 1 then press 5 1 508 383 7855 Contacting Technical Support C 3 To Reach Technical Support by Phone or Fax Outside N America Product Product Area PNA Custom and Synthesis Telephone 1 800 899 5858 press 1 then press 5 Fax 1 508 383 7855 FMAT 8100 HTS System CytoFluor 4000 Fluorescence Plate Reader 1 800 899 5858 press 1 then press 65 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U
31. TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS E 2 Limited Warranty Statement Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com AS Applied ww Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 06 2001 Part Number 4307577C an Applera business
32. TaqMan Human Cytokine Card Protocol Applied KS Biosystems Copyright 2001 All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with ir arising from the use of this document NOTICE TO PURCHASER DISCLAIMER OF LICENSE The TaqMan Human Cytokine Card is optimized for use in the Polymerase Chain Reaction PCR and 5 nuclease detection methods covered by patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd No license under these patents to use the PCR process or 5 nuclease detection methods is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the PCR process for certain research and development activities accompanies the purchase of certain Applied Biosystems reagents when used in conjunction with an authorized thermal cycler or is available from Applied Biosystems Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 9
33. an Total RNA 60 ng 2 ug Performing Reverse Transcription Guidelines Follow the guidelines below to ensure optimal RT performance The TaqMan Human Cytokine Card is designed to assay cDNA generated from only human total RNA samples Poly A RNA samples are not recommended for cytokine card experiments because most rRNA has been removed from them A 100 uL RT reaction will efficiently convert a maximum of 2 pg total RNA to cDNA Perform multiple RT reactions in multiple wells if using more than 2 yg total RNA Use only random hexamers to reverse transcribe the total RNA samples for cytokine gene expression assays Reverse Transcription 2 3 Preparing the The following procedure describes the preparation of three different test Reactions samples and a calibrator sample for reverse transcription Scale the recommended volumes accordingly for the number of samples needed using the TaqMan Reverse Transcription Reagents P N N808 0234 2 4 Reverse Transcription To prepare the reverse transcription reactions Step Action 1 In a1 5 mL microcentrifuge tube prepare a reaction mix for all total RNA samples to be reverse transcribed Volume pL Per Reaction Final Component Sample Mix x4 Value 10X RT Buffer 10 0 40 0 1X 25 mM MgCl 22 0 88 0 5 5 mM deoxyNTPs Mixture 20 0 80 0 500 yM per dNTP Random Hexamers 5 0 20 0 2 5 uM RNase Inhibitor 2 0 8 0 0 4 U
34. asurements There are two methods for removing outlying data from the relative quantification calculation Manual removal using the SDS software optional Manual removal using the Relative Quantification software recommended The following section explains the manual mode for removing outlying data using the SDS software Note The latter method is explained in the ABI PRISM 7700 Relative Quantification Software User s Manual Manual Removal of Outlying Data Using SDS Optional Visualizing To ensure precise relative quantification replicate groups must be Outliers carefully scrutinized for outlying wells The C vs Well Position view of the Amplification Plot allows you to examine each set of replicate wells for outliers To visualize the replicate groups for outlying amplification Step Action 1 From the Analysis menu select Amplification Plot 3 G The SDS software displays the results of the sequence detection run in an amplification plot of AR versus Cy From the Viewer pull down menu select Cy vs Well position J it Rn vs Cycles Viewer e aRn Baseline Subtracted Reporter The SDS software displays results as a C vs Well position plot 40 0 35 0 Cl Ihreshold Cycle S a 0 10 20 30 40 50 60 70 80 90 100 Well Position Select FAM from the Reporter pop up menu The SDS software displays the FAM Well versus Threshold Cycle
35. be separates the reporter dye and the quencher which results in increased fluorescence of the reporter Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye as shown below Polymerization R Reporter Q Quencher ome Qn O 3 5 5 Reverse Strand displacement 6 Primer d 3 S ss 5 F 3 Cleavage SS 3 3 5 3 V Polymerization completed y 3 e gt 5 5 m 3 5 3 lt E 5 5 When the probe is intact the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer F rster 1948 Lakowicz 1983 During PCR if the target of interest is present the probe specifically anneals between the forward and reverse primer sites The 5 3 nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target The probe fragments are then displaced from the target and polymerization of the strand continues The 3 end of the probe is blocked to prevent extension of the probe during PCR This process occurs in every cycle and does not interfere with the exponential accumulation of product The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR Because of these req
36. box of the Amplification Plot window Record the iph et stoe threshold a value Setting the Because the threshold value is only valid within a specific dye layer the Threshold for the FAM and VIC threshold values must be set independently FAM Dye Layer To set the threshold value for the FAM dye layer Step 1 Action Select FAM from the Reporter pop up menu in the Amplification Plot dialog box Viewer All TE Reporter ia The SDS software displays the FAM Amplification Plot Follow the procedure for Setting the Threshold for the VIC Dye Layer on pages 4 7 to 4 8 Click OK Example Amplification Curve The figure below shows a typical semi logarithmic FAM amplification plot with the correct threshold setting 101 100 ARn Amplification FAM Example JB TH WAN Cycle Number Data Analysis 4 9 Eliminating Outlying Amplification Overview For any PCR experimental error may cause some wells to amplify Modes of Outlier Removal 4 10 Data Analysis insufficiently or not at all These wells typically produce C values that differ significantly from the average for the associated replicate wells If included in the relative quantification calculations these outliers can potentially result in erroneous relative gene expression me
37. d D H 1991 Detection of specific polymerase chain reaction product by utilizing the 5 3 exonuclease activity of Thermus aquaticus DNA polymerase Proc Natl Acad Sci USA 88 7276 7280 Innis M A Myambo K B Gelfand D H and Brow M A 1988 DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA Proc Natl Acad Sci USA 85 9436 9440 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lakowicz J R 1983 Principles of Fluorescence Spectroscopy New York Plenum Press xiv 496 pp Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 References D 1 Limited Warranty Statement Applied Biosystems warrants to the customer that for a period ending on the earlier of one year from the completion of installation or fifteen 15 months from the date of shipment to the customer the Warranty Period the TaqMan Human Cytokine Card Upgrade Package purchased by the cu
38. display Data Analysis 4 11 Identifying To identify outlying replicate wells Outlying Replicate Wells Step Action 1 Verify the uniformity of each set of replicate wells by comparing the groupings of Cy values The figure below is an example CT vs Well profile The outlying wells have been circled to illustrate the point 40 0 8 30 0 O E 25 0 ot u i wat E 20 0 Fa E 15 0 0 10 20 30 40 50 60 70 80 90 100 Well Position Are outliers present Then Yes record the well numbers of all outlying wells No go to Exporting the Analyzed Run as a Results File on page 4 14 2 From the Reporter pop up menu select VIC The SDS software displays the C s for the endogenous control 3 Repeat step 1 for the VIC dye layer Click OK 4 12 Data Analysis Eliminating Wells To eliminate a well with invalid data from the relative quantification from the Analysis Calculation Step Action 1 Hold down the Shift key and click each outlying well identified on the previous page The SDS Software highlights each cell as it is selected 2 From the Sample Type pull down menu select Not In Use o Sample Type TARG Rell Target Thermal Cycler Conditions STND Standard Sample Name UNKN Unknown Replicate NTC No Template Control NAC No Amplification Control Quantity NPC No Probe Control
39. e A zero timepoint sample in a timecourse experiment An untreated sample versus treated samples A resting sample versus activated samples Note For more information on the use of a calibrator sample in relative quantification see ABI PRISM 7700 Sequence Detection System User Bulletin 2 Relative Quantitation of Gene Expression Introduction 1 7 Design Guidelines Observe the following guidelines when designing TaqMan Human 1 8 Introduction Cytokine Card experiments Load each card with sample specific PCR reaction mix CDNA sample TaqMan Universal PCR Master Mix 20X 18S Primer and Probe Mix made from a single sample Individual cards are not designed to evaluate multiple cDNA samples Install the Sequence Detection Systems SDS Software version 1 7 1 or later to all instruments devoted to running TaqMan Human Cytokine Cards Run TaqMan Human Cytokine Cards within 30 minutes of loading them with sample specific PCR reaction mix To ensure the highest degree of reproducibility Applied Biosystems recommends scheduling card runs so that each card is run within 30 minutes of the time it is loaded When a loaded card sits for an extended period of time the probes and primers within the wells of the card begin to diffuse into the adjoining channels This diffusion of critical reagents can diminish the potential signal generated during the PCR and can therefore affect the results of the experiment Run a
40. e types are incorrectly applied or are absent from the Sample Type palette a Add the sample types to the palette see Adding the TARG and ENDO Sample Types to the Dye Palette on page 6 11 b Apply the correct sample types to the SDS file c Re analyze and export the results Threshold values within the FAM and VIC dye layers may not be identical for all of the results files While viewing the card run within the SDS software verify the following The VIC threshold value must match the VIC thresholds for the other cards in the analysis The FAM threshold value must match the FAM thresholds for the other cards in the analysis If the threshold values are not consistent with the other cards in the analysis set them accordingly see Setting the Threshold Values on page 4 4 6 10 Troubleshooting Troubleshooting Interpreting Results continued Observation Possible Cause Recommended Action Average endogenous control Cys are 223 Not enough sample a Verify the type and quantity of the cDNA in the card sample loaded into the TaqMan Human Not enough cDNA from total RNA Cytokine Card See pages 3 4 to 3 5 for the correct template information b Increase the concentration of the cDNA sample loaded into the card Adding the TARG and ENDO Sample Types to the Dye Palette To add new dyes to the dye palette Step Action 1 From the S
41. eaa tad ae ee 2 6 3 PCR OVER VIC Ws e n chek s als dies ayaa hy aah A aE dat aaa E TE 3 1 Where You Are in the Procedure 000000005 3 1 Before Conducting the PCR 1 0 02 eee eee 3 2 Adjusting the Exposure Time for Card Runs 3 2 About the ABI PRISM Filling Station 0 000 000 0050 08 3 3 Descriptio Sens en a Sele eee bets we ae E ES 3 3 Laboratory Setup 0 0 eee eee eee eee 3 3 Sample Preparation 0 0 0 eee eee een eee 3 4 Guidelines oi ee i a E a E a idence E acs 3 4 Recommended Quantity 0 0 ee eee eee eee 3 5 Preparing a Sample Specific PCR Reaction Mix 3 5 Loading the TaqMan Human Cytokine Card 0 3 7 About ABI PRISM Cards 2 0 0 eee eee eee eee 3 7 Guidelines for Loading Cards 0 0 00 000000005 3 8 Preparing a Gard i 3053 4c ca oan Gaede Ate ae Ree Es 3 9 Loading and Sealing a Card 1 0 0 0 0 eee eee eee 3 12 Loading a Card into an ABI PRISM 7700 Sequence Detection System 3 14 About the ABI PRISM Card Adaptor Design 3 14 Loading a Card for Sequence Detection 3 15 Running TaqMan Human Cytokine Cards 000 3 17 About SDS Plate Documents 00 0000 000 3 17 Using a Template ac ies consist a aie aaa sl god ea eed Rae te Sed 3 17 Setting the Thermal Cycling Parameters 3 18 Configuring t
42. ed dU containing DNA by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil thereby creating an alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA Longo et al 1990 Please follow these recommended procedures Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated Maintain separate areas dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Use positive displacement pipets or aerosol resistant pipet tips Clean lab benches and equipment with 10 bleach solution Introduction 1 9 Materials and Equipment TaqMan Card The TaqMan Human Cytokine Card Upgrade Package and Upgrade Package components are available as follows and Components Components Part Number TaqMan Card Upgrade Package 4311899 ABI PRisM 7700 Relative Quantification Software 4313010 ABI PRism Card Adaptor ABI PRIsM Card Filling Station TaqMan Human Cont
43. elative Quantification of Gene Expression P N 4303859 ABI PRISM 7700 Relative Quantification Software User s Manual P N 4309937 Configuring the ABI PRISM 7700 Relative Quantification Software Creating a The ABI PRISM 7700 Relative Quantification Software User s Manual Template for will instruct you to create a template file for TaqMan Human Cytokine Card Analyses Card analyses The template file is time saving device that will allow you to skip the laborious task of repetitive plate setup and configuration The following figure has been included to provide you with an example of a template document with the correct target configuration and layout untitled Fell Information Plate Group Setup Actions Target Sample Target Ct Endogenous Ctrl Ct Target Dye Endogenous CtrIDye Calibrator earar Calibrator L 1apha L 1apha L 1apha L 1apha IL 1 beta IL 1beta IL 1betalIL 1beta IL 2 IL 2 IL 2 IL 2 Cabrator Cabrator Cabrator Cabrator Cabrator Cabrator Cabrator Cabrator Cabrator Cabrator Cabrator Calbrator IL 3 IL 3 IL 3 IL 3 IL IL 5 IL 5 IL 5 Calbrator Calbrator Cabrator Calibrator eee ator Calibre ator Calbrator Calibrator Calibrator L 4 ator Cabr IL 6 IL 6 IL 6 IL 6 IL IL 7 IL 8 IL 8 Cabrator Cabrator Cabrator Calbrator Calbr ator Cabrator Cabrator Calibr are Calbrator Calibrator IL 10
44. ells A1 A12 The alignment pins on the 7700 Sample Block may be interfering with heat transfer The pins of earlier instruments See Figure 6 5 can prevent the lens plate of the ABI PRISM Card Adaptor from aligning with rows A and B Use Figure 6 5 to determine whether the pins on your instrument must be replaced If your instrument does not contain the correct type of pins replace them with the new pins supplied in the TaqMan Card Upgrade Package REPLACE DO NOT REPLACE 2 2222622626226 2E226226262262262 22262222 22222 e eEeEeEGeE EEE Ee amp 2 2 Ee eE eE E E222 QO Figure 6 5 Pin Replacement GR1550 6 8 Troubleshooting Correcting for Plate Document Type To correct a mislabeled document Step Action 1 Save and Close the current plate document 2 From the File menu select New 3 N To correct a mislabeled document continued Step Action 3 Configure the new file as follows From menu Select Plate Type Single Reporter Plate Format The Card Run Real Time 4 From the Import submenu off of the File menu select Labview Format Raw Data New Plate Open Plate 0 Close sw Save 38 Save A
45. emical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Site Preparation A site preparation and safety guide is a separate document sent to all and Safety Guide customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order documents by automated telephone service 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request To order documents by telephone In the U S Dial 1 800 345 5224 and press 1 In Canada then 2 then 1 To order in English dial 1 800 668 6913 and press 1 then 2 then 1 To order in French dial 1 800 668 6913 and press 2 From any See the specific region under To Contact Technical other couniry Support by Telephone or Fax Outside North America To view download or order d
46. ems SDS instrument The card adaptor is a specialized device that ensures adequate heat transfer and fluorescent data collection occur during the PCR The following figure illustrates the components of the adaptor Lens plate Reaction card Reflective plate Component Description Lens plate Houses 96 lenses that direct the focal point of the argon ion laser into the wells of the reaction card The top of the lens plate contains a label to indicate the correct orientation of the plate Reaction card Contains a cDNA sample and the necessary reagents for the PCR Reflective plate An aluminum plate that ensures efficient conduction and uniform heat transfer to all wells of the reaction card The plate contains a notched corner to aid in correctly orienting the card for thermal cycling 3 14 PCR Loading a Card To load the card into the ABI PRISM 7700 Instrument for Sequence Detection Step Action 1 Slide the sample block cover back exposing the sample block 2 Place the reflective plate on top of the sample block so that the notched edge is located in the far right corner Notched corner 3 Place the filled TaqMan Human Cytokine Card on top of the reflective plate so that the keyed corner of the card aligns with the notched edge of the plate Notched corner of the plate Keyed corner of the card PCR 3 15 3 16 PCR To load the card into
47. equired for optimal AmpErase UNG enzyme activity b Required for AmpliTaq Gold DNA Polymerase activation c Asetting of 99 C is necessary for this step because of the ABI PRISM card s unique thermokinetic properties 6 Click the Sample Volume text field and type 100 pL 3 18 PCR Configuring the Dye Layers Note If the TARG RelQ Target or the ENDO RelQ Endogenous Control do not appear in the Sample Type pop up menus configure the sample types as explained on page 6 11 To configure the FAM and VIC dye layers Step Action 1 Select FAM from the Dye Layer pop up menu 2 Select all wells of the plate document 3 Select TARG RelQ Target from the Sample Type pop up menu The SDS software labels all selected wells as TARG Show Analysis Dye Layer Fat 4 Select VIC from the Dye Layer pop up menu 5 Select all wells of the plate document 6 Select ENDO RelQ Endogenous Control from the Sample Type pop up menu 7 Select cells A1 A4 PCR 3 19 To configure the FAM and VIC dye layers continued Step Action 9 Repeat steps step 7 8 for each target cytokine so that the plate document mirrors the assay configuration of the TaqMan Human Cytokine Card TaqMan Human Cytokine Card Assay Configuration 3 20 PCR Saving the Plate To save the plate document as a template file Docume
48. etup menu select Sample Type Palette The Sample Type Palette dialog box appears 2 Click Sample Type Setup The Sample Type Setup dialog box appears 3 Add the TARG RelQ Target sample type to the palette a Click Add A new row appears at the bottom of the dye list b Click the Acronym text field and type TARG Click here Ferca E e c Click the Name text field and type RelQ Target Click here mes Bere E wm 3 d Click the Color field When the Color pallet dialog box appears Select a color for the new dye and click OK Double click Rel Target rone here The color field for the new dye fills with the new color e From the Reporter pull down menu select FAM Fan Select FAM 4 Troubleshooting 6 11 6 12 Troubleshooting To add new dyes to the dye palette continued Step Action 4 Add the ENDO RelQ Endogenous Control sample type to the palette a Click Add A new row appears at the bottom of the dye list b Click the Acronym text field and type ENDO c Click the Name text field and type RelQ Endogenous Control d Click the Color field The Color pallet Dialog Box appears e Select a color for the new dye and click OK The color field for the new dye fills with the new color f From the Reporter pull down menu select VIC Select VIC enoo RelQ Endogenous Control Click OK The new sample types will now be available from the Sample Type pop up me
49. he Dye Layers 00 02 e eae 3 19 Saving the Plate Document as a Template 3 21 Running the Card ivi cet swie then led beh woe eee ee dae ed 3 22 4 Data Analysis QVEL VIEWS e E e sabe A A team whe aloes whe aid E iar 4 1 Where You Are in the Procedure 00 0002 4 1 Before the Analysis iessen opr sian esi eal e ee set eas 4 2 Activating Spectral Compensation 0 000000 4 2 Non Fluorescent Quencher 00 0 cece cee eee eee eee 4 3 Setting the Baseline Values 00 0 0 iaai ne eaa eee eee 4 3 Automatic Baseline Calculation 0 00 00 0 0000 4 3 Setting the Threshold Values 0 0 0 cece eee cee eee 4 4 Threshold Requirements for Relative Quantification 4 4 Displaying Results on an Amplification Plot 4 4 Threshold Value Basics 1 2 00 0 cc cece a nia ee eens 4 5 Guidelines for Setting Thresholds 000 4 6 Setting the Threshold for the VIC Dye Layer 4 7 Setting the Threshold for the FAM Dye Layer 4 9 Eliminating Outlying Amplification 0 00 00 02 0000 4 10 OVELVICW aoi See o oh see gle we Rear a Se ee ete a oes 4 10 Modes of Outlier Removal 2 0 2 0 0 00 0 c eee eee eee 4 10 Manual Removal of Outlying Data Using SDS Optional 4 11 Visualizing Outliers 2 0 6 eee eee 4 11 Identifying Outlying Replicate Wells
50. he next sample specific PCR reaction mix in the comparative experiment Repeat the PCR step for each of the remaining cDNA samples and then proceed to Chapter 4 Data Analysis 3 22 PCR Data Analysis Overview Where You Are in the Procedure You Are Here Before calculating relative quantification values from the results of the card run the raw data must be analyzed and exported to a results file The analysis procedure consists of setting threshold values for the FAM and VIC dye layers and eliminating outlying amplification Reverse Transcription cDNA generation PCR ABI Prism 7700 Card Run Data Analysis Performed using the Sequence Detection Systems Software a Activate Spectral Data Analysis Compensation Performed using the Sequence b Analyze the run data Detection Systems Software c Set Threshold Value for the VIC Dye Layer d Set Threshold Value for the FAM Dye Layer e Eliminate outlying Interpreting Results amplification Performed using the ABI Prism f Export the analyzed data 7700 Relative Quantification for relative quantification Data Analysis 4 1 Before the Analysis Activating Spectral Compensation 4 2 Data Analysis For all real time runs set the spectral compensation to ON during analysis of multiplex PCR assays Activating spectral compensation provides improved spectral resolution for multi reporter applications
51. hed to the lid at all times except during maintenance or transportation of the station Vacuum hose quick release valve Vacuum attachment Turn on the vacuum pump Allow the vacuum pump to evacuate the card until the gauge on the hose stabilizes at or less than 600 microns IMPORTANT Do not fill the card at a vacuum greater than 600 microns Above that reading the vacuum is not strong enough to adequately fill the card and may result in the loss of both the cDNA sample and the card Do not attempt to refill partially filled cards PCR 3 11 Loading and To fill and seal the card for thermal cycling Sealing a Card 3 12 PCR Step Action 1 After the vacuum reaches 600 microns pipet 300 pL of the sample specific PCR reaction mix CDNA sample TaqMan Universal PCR Master Mix 20X 18S Primer and Probe Mix from page 3 5 into the fill reservoir of the Filling Station Use the pipet tip to dislodge any bubbles that appear at the bottom of the fill reservoir In one motion firmly pull the Filling Station actuator to its maximum extension Note Pulling the actuator may be fairly difficult and require some physical strength The TaqMan Human Cytokine Card fills with sample specific PCR reaction mix IMPORTANT Do not attempt to force the actuator back into the closed position It retracts automatically when the lid is opened ae Pull Complete the following as quick
52. id transfer through the microchannels of the fill consumable Do not remove a card from its packaging until you are ready to load it with sample specific PCR reaction mix Excessive exposure to light damages the fluorescent probes Do not twist or bend the soft fill consumable The seal between the reaction card and the fill consumable is crucial to the loading procedure If broken the seal may leak and result in an inadequately filled card Use caution when opening and closing the ABI PRISM Filling Station The station lid is not designed to remain in an open position NAN PHYSICAL HAZARD If left open the ABI Prism Filling Station lid may unpredictably close and cause an injury Preparing a Card PNW PHYSICAL HAZARD If left open the ABI Prism Filling Station lid may unpredictably close and cause an injury The lid is not designed to remain in an open position To prepare the TaqMan Human Cytokine Card for loading Step Action 1 Carefully remove a TaqMan Human Cytokine Card from its packaging Fold the adhesive flap attached to the card back in order to accommodate the fill consumable Adhesive flap Attach a fill consumable to the card a Remove the white adhesive backing from the fill consumable b Align the protruding aperture and two pins on the edge of the fill consumable to the holes in the card see below c Once aligned press gently on across both sides to secure the f
53. ill consumable in place Aperture PCR 3 9 3 10 PCR To prepare the TaqMan Human Cytokine Card for loading continued Step Action 4 Carefully load the card into the ABI PRISM Card Filling Station a Orient the card so that the pins on the station align with the holes in the soft fill consumable as indicated in the figure below b Once the pins are correctly aligned press down firmly on the top of the fill consumable to ensure a good fit IMPORTANT Do not press down on the junction between the fill consumable and cytokine card IMPORTANT The adhesive flap must be folded away from the fill consumable for proper operation of the Filling Station The following illustration demonstrates the correct technique for loading a card into the Filling Station away from the fill consumable Fit the card s fill consumable alignment holes to the pins in the Filling Station To prepare the TaqMan Human Cytokine Card for loading continued Step Action 5 Close the ABI PRISM Card Filling Station lid IMPORTANT Press firmly on the top of the ABI PRISM Card Filling Station to ensure that it is completely closed U Trerisoe If necessary connect the vacuum hose to the attachment on the ABI PRISM Filling Station lid The end of the vacuum hose contains a quick release valve that clicks when locked into place Note Leave the vacuum hose attac
54. into a TaqMan Human Cytokine Card c Load the filled card into the ABI PRism 7700 Instrument Data Analysis d Create a plate document Performed using the Sequence template for TaqMan Detection Systems Software Human Cytokine Card runs OR If a template file exists create a plate document Interpreting Results from the template Performed using the ABI Prism e Begin the PCR sequence 7700 Relative Quantification detection run PCR 3 1 Before Conducting the PCR Adjusting the ABI PRISM cards typically return a stronger fluorescent signal than Exposure Time for standard MicroAmp Optical Plates Consequently the CCD camera Card Runs exposure time must be decreased to compensate for the higher signal intensity Step Action 1 Launch the Sequence Detection Systems SDS software 2 Select Advanced options from the Diagnostics submenu off of the Instrument menu 3 From the Miscellaneous group box activate the manual setting a Click the Set 7700 Exposure Time checkbox b Click the Set 7700 Exposure Time for Cards text field and type 10 Advanced Options Yiewer E Display mse in Multicomponent Yiew C Display best fit in Raw Spectra View Analysis Spectra Components Oo Use background in Spectra Components folder Oo Use pure spectra in Spectra Components folder Miscellaneous Click here Fa Set 7700 Exposure Time for Plates for Ca
55. ion Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation NIA Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices NATIT Indicates a potentially hazardous situation which if not avoided could result in death or serious injury 7 lelsts Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations 7 NAVE Tite CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS 4 Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Check regularly for ch
56. is performing properly if the average VIC Cis lt 12 cycles Amplification TaqMan Human Total RNA VIC 0 10 20 30 40 50 60 70 80 90 100 Well Position Demonstrating Performance with Control RNA B 3 To verify the C values continued Step Action 7 Threshold Cycle Ct pe oO O From the Reporter menu select FAM The card is performing properly if the following FAM C s are lt 30 cycles Cytokine Target Wells IL 10 37 40 LT B 81 84 TGF B 85 88 TNF a 89 92 TNF B 93 96 Note The TaqMan Human Total RNA sample expresses some cytokines other than those listed above to a limited extent Amplification TaqMan Human Total RNA VIC Well Position Export the analyzed data as explained in Exporting the Analyzed Run as a Results File on page 4 14 B 4 Demonstrating Performance with Control RNA Verifying the Expression Profile of Control RNA Transferring Data from the Results File Note For the purpose of demonstration the following procedure illustrates how to generate a profile of median AC values using Microsoft s Excel spreadsheet utility Other software packages can be used to create the profile however the keystrokes and command pathways may differ After exporting the results of the control card run the performance of the card can be verified again by generating a profile of the median AC value
57. ision of the data see Figure 6 3 Amplification Plot AR vs Cycle 10 Samples Mi 100 M be r Zj 10 1 bi Ej zj 10 2 Mi Mi Viewer aaa 2 39 Reporter MC T Slow upward slope during the later cycles 15 35 Downward slope during the early cycles 1 10 Figure 6 3 Incorrect Plate Format 6 6 Troubleshooting Troubleshooting Data Analysis continued Observation Possible Cause Recommended Action Some growth curves The instrument is Correcting the problem is optional slope down toward the becoming saturated later cycles of large growth curves Figure 6 4 The problem cannot be corrected for the current run To avoid this problem in future runs follow the procedure Adjusting the Exposure Time for Card Runs on page 3 2 Amplification Plot AR vs Cycle 2 000 1 800 1 600 Li l 1 400 4 ST eB Ltt pw 1 200 44 aS 1 000 AR 0 800 0 600 IBIS ee t Downward sloping growth curves v 400 J t t t 1 a dl NT Md He S ya MOL ozoo HHH Hte e E tt U 0UU 0 200 Cycle Figure 6 4 Instrument Saturation Troubleshooting 6 7 Troubleshooting Data Analysis continued Observation Possible Cause Recommended Action Poor to no amplification in the wells of the top row w
58. ll TaqMan Human Cytokine Cards from the same comparative experiment on the same ABI PRISM 7700 instrument Running all cards from the same experiment on one ABI PRISM 7700 instrument ensures a high degree of reproducibility and consistency When analyzing results with the SDS software Set FAM dye layer threshold values identically for all cards in the same comparative experiment Set VIC dye layer threshold values identically for all cards in the same comparative experiment To compare the data from different card runs the threshold values for each card must match exactly The ABI PRISM 7700 Relative Quantification Software cannot analyze data from card runs that have different threshold values within the respective dye layers Preventing Contamination Contamination and the 5 Nuclease Assay Using AmpErase UNG General PCR Practices PCR using the 5 nuclease assay requires special laboratory practices to avoid false positive amplifications Kwok and Higuchi 1989 The assay s high throughput repetitive nature can potentially amplify a single DNA molecule Saiki et al 1985 Mullis and Faloona 1987 AmpErase uracil N glycosylase UNG is a pure nuclease free 26 kDa recombinant enzyme encoded by the Escherichia coli uracil N glycosylase gene The gene was inserted into an E coli host to direct expression of the native form of the enzyme Kwok and Higuchi 1989 UNG acts on single and double strand
59. lts window The following figure illustrates a typical TaqMan Human Cytokine Card profile Legend E E sirp 2 EDE Sample 4 Target 10 Target 11 Target 12 Target 13 Target 14 Target 15 Target 16 Target 17 Target 18 Target 19 Target 20 Target 23 Target Dye F AM x Not enough sigma te calculate ACH reliably Endogenous Control Dye VIC Value disatayed is a minimam Calibrator Sample Sample 1 Kave dispisyed amp a maximun X Axis The X axis of the gene expression profile lists all of the cytokine targets Y Axis involved in the analysis Within each grouping the software displays the relative quantity of the target in each sample The Y axis displays the relative quantities of the cytokine targets on a semi logarithmic scale The quantities shown are relative to the calibrator sample Each increment corresponds to a ten fold difference in gene expression Note Because the calibrator sample is compared to itself the level of CDNA expression in the calibrator always appears as 1 1E 00 Interpreting Results 5 5 Sample Bars Each bar represents the level of target gene expression within a sample listed in the legend 5 6 Interpreting Results Symbol Definition 7 Minimum target gene expression level Displayed on all associated sample bars when A calibrator sample produces an Average C
60. ly as possible a Turn off the vacuum pump b Open the Filling Station lid NYG Me PHYSICAL HAZARD If left open the ABI PRISM Filling Station lid may unpredictably close and cause an injury The lid is not designed to remain in an open position Remove the card from the Filling Station To fill and seal the card for thermal cycling continued Step Action 5 Detach and discard the fill consumable then remove any adhesive remaining on the surface of the card IMPORTANT Remove all of the adhesive from the card The adhesive may interfere with the card seal and allow the sample specific PCR reaction mix to leak during thermal cycling 62 gt Sere ms Ow Ler e722 oe oe oe Le a e o oD PSE E E a 2 a 2 Oo ON 2 2 2e or KEE SL a2 a2 a 2 ww SS oe Sle oCo at N gt 2 52 9 gt 2 _ 2 eo o KN SF PF De ma G Roe oF Q fe 4 lt a gt lt Note NSS ta gt V 6 7 Fold the adhesive flap over the front edge of the card and press firmly on the flap to ensure an adequate seal The card is now filled and ready to be loaded into the ABI PRISM 7700 Sequence Detection System PCR 3 13 Loading a Card into an ABI PRISM 7700 Sequence Detection System About the The unique design of the TaqMan Human Cytokine Card requires the ABI PRISM Card use of an ABI Prism Card Adaptor for use on the ABI PRISM 7700 Adaptor Design Sequence Detection Syst
61. mable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Limited Warranty Statement E 1 Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems sole expense No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contained in Applied Biosystems printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems will not be binding on Applied Biosystems Applied Biosystems shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties This Warranty is limited to the original purchaser and is not transferable THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS
62. manual Chapter 3 PCR Conduct the data analysis as described in Appendix B Demonstrating Performance with Control RNA Designing TaqMan Human Cytokine Card Experiments About the Comparative Cry Method of Relative Quantification Significance of the Calibrator Sample Relative gene expression values can be obtained from ABI PRISM 7700 run data using the Comparative C Method for relative quantification In the Comparative C Method quantity is expressed relative to a calibrator sample that is used as the basis for comparative results Therefore the calibrator is the 1X sample and all other quantities are expressed as an n fold difference relative to the calibrator For more information on the Comparative C Method of Relative Quantification see the following publications ABI PRISM 7700 Sequence Detection System User Bulletin 2 Relative Quantitation of Gene Expression ABI PRISM 7700 Relative Quantification Software User s Manual All TaqMan Human Cytokine Card relative quantification experiments require data from a calibrator sample During analysis the ABI PRISM 7700 Relative Quantification Software calculates the levels of cytokine gene expression in samples relative to the level of expression in the calibrator Thus the calibrator sample is an integral part of the relative quantification calculation because it serves as the basis for the comparative results Examples of possible calibrator samples includ
63. med using the ABI PRISM 7700 Relative Quantification Reverse Transcription 2 1 Preparing the RNA Template Recommended Use only human total RNA samples to generate cDNA for the TaqMan Human Cytokine Card Template The following table lists the known template incompatibilities Template Explanation Poly A The 18S rRNA endogenous control assay cannot accurately evaluate cDNA generated from poly At RNA samples because most of the rRNA has been removed from them Non human Except for 18S rRNA all assays on the TaqMan Human Cytokine Card are human specific Template Quality The quality of your results is directly related to the purity of your RNA template Therefore use only well purified samples with the TaqMan Human Cytokine Card Because ribonuclease and genomic DNA contamination are common problems in gene expression studies purify your samples accordingly to ensure the best results Template Quantity 2 2 Reverse Transcription Note TaqMan Human Cytokine assays have been experimentally proven not to detect up to 10 000 copies of contaminating genomic DNA per card If possible use spectrophotometric analysis to determine the concentrations of purified total RNA samples before reverse transcription The table below lists the recommended range of initial template quantities for the reverse transcription RT step Initial Template Quantity of total RNA per 100 yL RT reaction Hum
64. ne expression levels from the threshold cycle C values obtained from TaqMan Human Cytokine Card runs The C is the cycle at which the first statistically significant increase in AR is detected during the PCR Wells with greater initial template concentrations reach their C values at lower cycle numbers than wells containing lower template concentrations Because amplicons designed and optimized according to Applied Biosystems guidelines have amplification efficiencies approaching 100 each PCR cycle corresponds to a twofold increase in product Similarly a change in C value of one cycle equates to a twofold difference in initial template concentration The relationship between C value and initial template concentration is the basis of the relative quantification calculation The software calculates relative levels of gene expression by comparing the normalized cytokine assay C s of experimental samples to a calibrator sample The software displays the calibrator as a 1X sample and all other quantities as an n fold difference relative to the calibrator Note For more information about Applied Biosystems guidelines for amplicon design see the TaqMan PCR Universal Master Mix Protocol P N 4304449 For more information on the topic of the Comparative C Method for relative quantification of gene expression Applied Biosystems recommends the following resources ABI PRISM 7700 Sequence Detection Systems Software User Bulletin 2 R
65. ner Mass Spectrometers Voyager Mass Spectrometers MassGenotyping Solution 1 MGS1 System LC MS Applied Biosystems MDS Sciex Chemiluminescence Tropix support sciex com tropix appliedbiosystems com To Reach Technical Support by Phone or Fax N America To contact Applied Biosystems Technical Support in North America use the telephone or fax numbers in the table below Note To schedule a service call for other support needs or in case of an emergency dial 1 800 831 6844 then press 1 Product Product Area Telephone Fax ABI PRISM 3700 DNA Analyzer 1 800 831 6844 then press 8a 1 650 638 5981 DNA Synthesis Fluorescent DNA Sequencing 1 800 831 6844 press 2 then press 1a 1 800 831 6844 press 2 then press 2a 1 650 638 5981 1 650 638 5981 Fluorescent Fragment Analysis including GeneScan applications 1 800 831 6844 press 2 then press 3a 1 650 638 5981 Integrated Thermal Cyclers ABI PRisM 877 and Catalyst 800 instruments 1 800 831 6844 press 2 then press 42 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer 1 800 831 6844 press 2 then press 62 1 650 638 5981 C 2 Contacting Technical Support Product Product Area Peptide Synthesis 433 and 43x Systems Telephone 1 800 831 6844 press 3 then press 12 Fax 1 650 638 5981 Protein Sequencing Procise Protein Sequencing
66. nt as a Template Step Action 1 Select Save As from the File menu The Save As dialog box appears 2 Complete the following actions a Click the Stationery Pad radio button b Click the Save this document as text field and type a name for the template file a Earth Cytokine Plate Template Eject Save this document as bs Click here Cytokine Plate Template Click here and type a file name 3 Select a location for the software to place the template file Click OK The software saves the template file 5 Select Close from the File menu When prompted to save the document select Don t Save The software closes the template document PCR 3 21 Running the Card To run the card using the template file Step Action 1 Select Open from the File menu 2 Using the browser select the Cytokine Card Template file 3 Click Open The SDS software creates a plate document with attributes identical to that of the template file Click the Show Analysis button to toggle to the Analysis View 5 Click Run to begin thermal cycling Note Ifa card is run immediately after a plate the instrument may pause momentarily before ramping The unique thermokinetic properties of the TaqMan Human Cytokine Card require that the instrument cool the heated cover to 65 C before initiating thermal cycling When the first card run is complete prepare load and run t
67. nu Theory of Operation Overview This appendix provides an overview of the theoretical basis for the TaqMan Human Cytokine Card chemistry and ABI PRISM 7700 Sequence Detection System data collection Theory of Operation A 1 TaqMan Human Cytokine Card Chemistry RT PCR Basics of the 5 Nuclease Assay A 2 Theory of Operation The TaqMan Human Cytokine Card evaluates gene expression in a two step reverse transcription polymerase chain reaction RT PCR The figure below illustrates the assay steps Extension of primer on mRNA 5 3 mRNA RT irom cDNA Step Hexamer Synthesis of 1st cDNA strand 3 5 cDNA Extension of primer on cDNA 3 5 gt Forward oO Primer amp Synthesis of 2nd cDNA strand PCR 3 5 Step 5 3 PCR amplification of cDNA N i Forward Primer 5 2 gh S gt O 5 a ise Reverse x Primer 0 In the RT step cDNA is reverse transcribed from total RNA samples using random hexamers from the TaqMan Reverse Transcription Reagents In the PCR step products are synthesized from cDNA samples using the TaqMan Universal PCR Master Mix and target specific primers probes The PCR reaction exploits the 5 nuclease activity of AmpliTaq Gold DNA Polymerase to cleave a TaqMan probe during PCR The TaqMan probe incorporates a reporter dye VIC or FAM at the 5 end of the probe and a quencher TAMRA or MGB at the 3 end of the probe During the reaction cleavage of the pro
68. ocuments through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page click Documents on Demand then click MSDS Click MSDS Index search through the list for the chemical of interest to you then click on the MSDS document number for that chemical to open a pdf of the MSDS For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Introduction 1 13 Reverse Transcription Overview Where You Are in the Procedure You Are Here Synthesis of cDNA from total RNA samples is the first step in the two step RT PCR cytokine gene expression quantification experiment In this step random hexamers from the TaqMan Reverse Transcription Reagents prime total RNA samples for reverse transcription using MultiScribe Reverse Transcriptase Reverse Transcription Reverse Transcription cDNA generation cDNA generation a Prepare the reverse transcription reaction mix b Dilute total RNA samples PCR c Aliquot the reaction mix to ABI Prism 7700 Card Run each dilute total RNA sample d Load the samples onto a thermal cycler and program it with the RT Data Analysis conditions Performed using the Sequence e Begin reverse Detection Systems Software transcription thermal cycling Interpreting Results Perfor
69. ort is damaged If the problem persists replace the O ring on the top plate of the card adaptor with the part included with the kit The alignment pins on the 7700 Sample Block may be interfering with heat transfer See Poor to no amplification in the wells of the top row wells A1 A12 on page 6 8 AR So lo l i Amplification Plot AR vs Cycle Samples Viewer 30 Cycle Figure 6 1 Little or No Growth in the Amplification Plot Multicomponent View 2500 35 Reporter 20007 15004 Component Series Hi B E4BKGND C7 Hi Fluorescent Intensity a So Q O T T 200 300 400 500 600 700 Reading Figure 6 2 Degrading Multicomponent Signals Troubleshooting 6 5 Troubleshooting Data Analysis continued Observation Possible Cause Recommended Action Growth curves from The plate document file Create a new plate document for a card run and wells without is in Standard Plate import the Labview file from the problem run amplification drift format See Correcting for Plate Document Type on upward during the later page 6 8 cycles 15 35 see Figure 6 3 AND OR Note The poor precision of the problem run cannot be corrected after the run is complete Growth curves on all wells slope downward during the early cycles correlating with a loss in prec
70. ot contain fluorescence data from a sequence detection systems run Once created an unlimited number of identical SDS plate documents can be created from the template Because comparative analysis involves multiple runs and SDS plate documents for cards are identical it is more efficient to create and use a template file than to create a plate document for each card run PCR 3 17 Setting the To configure the PCR thermal profile for the reverse transcription Thermal Cycling Parameters Step Action 1 Launch the Sequence Detection Systems software Note If a plate document automatically appears on your screen select Close from the File menu to close it From the File menu select New 36 N Configure a new plate document with the following attributes From menu Select Plate Type Single Reporter Plate Format The Card Run Real Time Note TaqMan Human Cytokine Cards can only be used with SDS software version 1 7 1 or later If the options above do not appear in the New Plate dialog box update your instrument software 4 Click the Thermal Cycler Conditions button 5 Configure the thermal cycling profile with the following conditions Stage 1 2 3 Description UNG AmpliTag Gold PCR Activation Activation gt HOLD HOLD CYCLE 35 cycles Denature Anneal Extend Temperature 50 C 99 Cc 99 Cc 60 C Time 2 min 10 min 15 sec 1 min a R
71. pL MultiScribe Reverse 6 25 25 0 3 125 U L Transcriptase 50 U uL Total 65 25 261 0 a If changing the reaction volume make sure the final proportions are consistent with the recommended values above 2 Label four 1 5 mL microcentrifuge tubes for the three test samples and the calibrator sample 3 Transfer 60 ng 2 ug up to 34 75 uL of each total RNA sample to the corresponding microcentrifuge tube 4 If necessary dilute each total RNA sample to a volume of 34 75 uL with RNase free deionized water Cap the tubes and gently tap each to mix the diluted samples Briefly centrifuge the tubes to eliminate air bubbles in the mixture Label four 0 2 mL MicroAmp Reaction Tubes for the three total RNA test samples and the calibrator sample To prepare the reverse transcription reactions continued Step Action 8 Pipet 65 25 pL of the reaction mix from step 1 to each MicroAmp Reaction Tube from step 7 10X RT Buffer MgCl dNTPs Mixture Random Hexamers MultiScribe Reverse Transcriptase _ RNase Inhibitor 65 25 uL 65 25 uL 65 25 uL 65 25 uL Calibrator Sample 1 Sample 2 Sample 3 9 Transfer 34 75 pL of each dilute total RNA sample to the corresponding MicroAmp Reaction Tube 10 Cap the reaction tubes and gently tap each to mix the reactions 11 Briefly centrifuge the tubes to force the solution to the bottom and to eliminate air bubbles from the mixture Re
72. rdering In addition to the reagents supplied in the TaqMan Human Cytokine Card Upgrade Package other items are required for this protocol Unless otherwise noted many of the instruments and materials listed below are available from major laboratory suppliers MLS User Supplied Instruments Instruments Source ABI PRISM 7700 Sequence Detection System Applied Biosystems Microcentrifuge MLS Welch DUOSEAL Series Two Stage Belt Drive VWR Catalog Vacuum Pump P N 54973 075 Vacuum Trap Kontes VWR Catalog P N KT926300 0021 a Contact your local Applied Biosystems Sales Office for the instrument best suited to your needs See the back cover of this protocol for office locations b Substitute vacuum pumps must be oil based and capable of pulling a minimum vacuum of 2 5x10 3 Torr 600 microns User Supplied Materials Materials Source MicroAmp Reaction Tubes with Caps 0 2 mL Applied Biosystems P N N801 0612 Gloves disposable powder free MLS Microcentrifuge tubes sterile 1 5 mL MLS Pipettors positive displacement or MLS air displacement Pipette tips aerosol resistant MLS Polypropylene tubes MLS Water RNase free distilled deionized MLS Introduction 1 11 Safety Documentation User Attention Words Chemical Hazard Warning 1 12 Introduction Five user attention words appear in the text of all Applied Biosystems user documentat
73. rds Then click here Use Spectral Compensation for Real Time and type 10 Use Spectral Compensation for Endpoint Reference ROK 4 Click OK 3 2 PCR About the ABI PRISM Filling Station Description Samples are loaded individually into TaqMan Human Cytokine Cards for PCR thermal cycling using a specialized tool called the ABI PRISM Card Filling Station The Filling Station coordinates loading by first exposing the channels of the reaction card to a vacuum and then opening the evacuated card to a sample specific PCR reaction mix i EL i 2 o o o o m n ABI PRISM Card Filing Station Ss GR1696 S Number Component Description 1 Vacuum The attachment for connecting the Filling Attachment Station to a vacuum pump 2 Sealing Screw IMPORTANT Do not adjust The Filling 3 Actuator Screws Station screws are extremely sensitive 4 Actuator The switch for exposing the card to the PCR sample or the vacuum Laboratory Setup The ABI PRISM Filling Station operates in combination with a vacuum pump a vacuum trap and a gauge The figures below illustrate the arrangement of the equipment as seen in the laboratory PUMP VACUUM TRAP ABI PRISM 7700 FILLING VACUUM STATION GAUGE 5 Sample Preparation 3 4 PCR Guidelines Follow the guidelines below to ensure optimal PCR performance
74. re is a limit of five documents per fax request Obtaining Documents Through the Web Site To view download or order documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Documents on Demand 3 In the search form enter and select search criteria then click Search at the bottom of the page 4 In the results screen do any of the following Click the pdf icon to view a PDF version of the document Right click the pdf icon then select Save Target As to download a copy of the PDF file Select the Fax check box then click Deliver Selected Documents Now to have the document faxed to you Select the Email check box then click Deliver Selected Documents Now to have the document PDF format e mailed to you Note There is a limit of five documents per fax request but no limit on the number of documents per e mail request To Obtain To obtain Applied Biosystems training information Customer Training Information Step Action Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Training Contacting Technical Support C 7 References Forster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Annals of Physics Leipzig 2 55 75 Holland P M Abramson R D Watson R and Gelfan
75. rescent fluctuations due to changes in concentration or volume Normalization Normalization is accomplished by dividing the emission intensity of the reporter dye by the emission intensity of the Passive Reference to obtain a ratio defined as the R normalized reporter for a given reaction tube Variable Description Rat The R value of a reaction containing all components including the template R The R value of an unreacted sample This value may be obtained from the early cycles of a Real Time run those cycles prior to a detectable increase in fluorescence This value may also be obtained from a reaction not containing template AR The difference between the R value and the R value It reliably indicates the magnitude of the signal generated by the given set of PCR conditions The following equation expresses the relationship of these terms where n R emission intensity of reporter PCR with template emission intensity of passive reference Rn emission intensity of reporter PCR without template or emission intensity of passive reference early cycles of a Real Time reaction Theory of Operation A 5 Real Time The threshold cycle or C value is the cycle at which a statistically Detection significant increase in AR is first detected Threshold is defined as the average standard deviation of R for the early cycles multiplied by an adjustable factor On the
76. rming properly if the designated targets achieve the following AC values Target AC IL 10 Below 20 Lymphotoxin B Below 20 TGF B Below 17 TNF o Below 20 TNF B Below 17 B 8 Demonstrating Performance with Control RNA Contacting Technical Support Technical Support Contacting Technical Support To Reach Technical Support by E Mail You can contact Applied Biosystems for technical support By e mail By telephone or fax Through the Applied Biosystems web site You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems web site Please see the section To Obtain Technical Documents following the telephone information below To contact Applied Biosystems Technical Support by e mail for help in the following product areas Product Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and pcrlab appliedbiosystems com PCR Protein Sequencing Peptide and corelab appliedbiosystems com DNA Synthesis Contacting Technical Support C 1 Product Product Area E mail address Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems FMAT 8100 HTS System CytoFluor 4000 Fluorescence Plate Reader tsupport appliedbiosystems com Mari
77. rol Total RNA 50 ng 1 pL 4307281 TaqMan Human Cytokine Cards 10 cards 4330448 TaqMan Human Cytokine Card Protocol 4307577 TaqMan Universal PCR Master Mix 4304437 Vacuum tubing and gauge a Includes all the components listed above and a service installation visit b The TaqMan Universal PCR Master Mix is 2X in concentration and contains sufficient reagent to perform 33 cards 150 pL each The mix is optimized for TaqMan reactions and contains AmpliTaq Gold DNA Polymerase AmpErase UNG dNTPs with dUTP Passive Reference and optimized buffer components Storage Guidelines The table below lists the storage conditions for the kit materials Component Storage Conditions TaqMan Human Cytokine Cards 2 8 C dark 20X 18S Primer and Probe Mix 15 to 25 C TaqMan Universal PCR Master Mix 2 8 C dark TaqMan Human Control Total RNA 15 to 25 C TaqMan Human Control cDNA 15 to 25 C IMPORTANT Do not remove TaqMan Human Cytokine Cards from the packaging until ready to load them with reaction mix Excessive exposure to light can damage the probes 1 10 Introduction Ordering Applied Biosystems Kits and Reagents Reagents and Equipment Not Included To order additional kits and reagents please contact Applied Biosystems at one of the regional sales offices listed on the back of this protocol Have the part number of the kit or reagent of interest available when o
78. s and primers within the TaqMan Human Cytokine Card is performed as part of the Applied Biosystems manufacturing quality control process In this process the performance of each cytokine target assay is verified using plasmids that contain the cytokine s specific cDNA target sequence Introduction 1 5 System TaqMan Human Control Total RNA is available from Applied Biosystems Performance for demonstrating the performance of the TaqMan Human Cytokine Guarantee Card If the control total RNA is run in the card using the conditions 1 6 Introduction below the average C value for the 18S endogenous control will be fewer than 12 cycles and the AC values for five cytokine targets will be as follows Target ACA IL 10 Below 20 Lymphotoxin B Below 20 TGF B Below 17 TNF a Below 20 TNF B Below 17 a AC Median C FAM C VIC C for a group of replicates Note The targets above were chosen because they are significantly expressed in the control sample To achieve the above results follow the protocol in Chapter 2 Reverse Transcription to perform an RT conversion using 2 ug of control total RNA in 100 pL of reaction volume After the RNA is converted to cDNA make a sample specific PCR reaction mix using 150 pL Universal Master Mix 118 pL water 30 uL of 20X 18S Primer and Probe Mix and 2 pL CDNA Fill and run a TaqMan Human Cytokine Card with the reaction mix according to the procedure in this
79. s using a spreadsheet utility Because the control RNA sample lacks a calibrator sample for comparison relative quantities cannot be derived from the results of the control card run Instead the data from the control run can be used to generate a profile of the normalized median C values for the cytokine assays To transfer data from the results file to the spreadsheet Step Action 1 Launch Microsoft Excel 2 Open the results file from the control card run a From the File menu select Open The Open dialog box appears b Use the browser to navigate to and select the results file from the control card run c Click Open The software displays the data within the SDS results file 3 From the File menu select New A new spreadsheet appears 4 From the Window menu select the SDS results file The cytokine card results spreadsheet reappears Select cells A1 A97 From the Edit menu select Copy 7 From the Window menu select the new spreadsheet The new spreadsheet file reappears Click cell A1 9 From the Edit menu select Paste Excel pastes the data into the new spreadsheet Demonstrating Performance with Control RNA B 5 To transfer data from the results file to the spreadsheet continued Step Action 10 Repeat steps 3 7 to copy the FAM C values in cells F1 F97 from the results file and paste them into cell B1 B97 of the new file c D E F
80. stomer the Instrument will be free from defects in material and workmanship and will perform in accordance with the installation specifications set forth in the ABI PRISM 7700 and TaqMan Card Upgrade Installation Manual the Specifications During the Warranty Period if the Instrument s hardware becomes damaged or contaminated or it the Instrument otherwise fails to meet the Specifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense However if the ABI PRiISM Card Filling Station or Card Adaptor become damaged or contaminated or if the performance of the Instrument otherwise deteriorates due to consumables and or reagents other than those supplied or expressly recommended by Applied Biosystems Applied Biosystems will return the Instrument to Specification at the customer s request and at the customer s expense After this service is performed coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period This Warranty does not extend to any Instrument or part which has been a the subject of an accident misuse or neglect b modified or repaired by a party other than Applied Biosystems or c used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consu
81. t 1 8 Preventing Contamination 0 0 00 eee eee eee eee 1 9 Contamination and the 5 Nuclease Assay 0 000 005 1 9 Using AmpErase UNG 0 0 ee eee eee 1 9 General PCR Practices cse cocca esis eee eee eee 1 9 Materials and Equipment 0 0 00 cece eee eee eee 1 10 TaqMan Card Upgrade Package and Components 1 10 Storage Guidelines nnsa eee i ea aa ee eee eee 1 10 Ordering Applied Biosystems Kits and Reagents 1 11 Reagents and Equipment Not Included 1 11 Safety siete ee Rae Mle sea Steams RASS San AO Sen eS 1 12 Documentation User Attention Words 4 1 12 Chemical Hazard Warning 0 e cece eee eee eee 1 12 Site Preparation and Safety Guide 0004 1 13 Ordering MSDSs 0 0 eee cee eee eens 1 13 2 Reverse Transcription DVELVIC Wo sists aR AA a yale fy path a aaO dat aaa pica ant bleades c 2 1 Where You Are in the Procedure 000000005 2 1 Preparing the RNA Template 00 00 c eee eee eee eee 2 2 Recommended Template 0 0 cece eee eee eee eee 2 2 Template Quality 0 0 a eens 2 2 Template Quantity 2 2 eee eee 2 2 Performing Reverse Transcription 0 0 0 0 cece eee ee ee 2 3 GUISES ce ete brie gts swig E IS Ry dspace hee Met ged aS eI a 2 3 Preparing the Reactions 0 00 0 eee eee eee eee 2 4 Thermal Cycling wo cc seater y
82. ted from human total RNA in a two step RT PCR experiment The card functions as the reaction vessel for the PCR sequence detection step The wells of the card contain the fluorogenic 5 nuclease assays that detect the amplification of 24 cytokine targets Relative Introduction 1 1 TaqMan Human Cytokine Card Design 1 2 Introduction levels of cytokine gene expression are determined from the fluorescence data generated during PCR using the ABI PRism 7700 Relative Quantification Software The TaqMan Human Cytokine Card consists of a specially developed 96 well consumable divided into 24 sets of replicates one set for each cytokine assay Each well contains TaqMan MGB probes and primers for one human cytokine mRNA target A 20X 18S rRNA endogenous control of TaqMan MGB probe and primers is supplied for multiplex assays The figure below illustrates the configuration of the cytokine gene expression assays on the card IL 12p35 IL 15 G CSF IFN y TNF a 10 11 IL 2 IL 5 IL 8 IL 12p40 IL 17 GM CSF LT B TNF B 12 Procedure The following diagram provides an overview of this protocol Flowchart Reverse Transcription cDNA generation a Prepare the reverse transcription reactions b Thermal cycling PCR ABI PRISM 7700 card run sequence detection Prepare a sample specific PCR reaction mix Load a TaqMan Human Cytokine Card Program the ABI PRISM 7700 instrument Run the card
83. tokine Assays TaqMan probes and primers for the 24 cytokine target assays span exon junctions to minimize the contribution of contaminating genomic DNA Performance tests demonstrate that TaqMan assays can be run with samples containing up to 10 000 copies of genomic DNA without detection of contaminants TaqMan 18S rRNA Endogenous Control Assay The 18S rRNA endogenous control assay is not RNA specific and consequently is affected by genomic DNA contamination However because of the extremely high expression level of rRNA even gross contamination has a negligible effect on the relative quantification values obtained from the card Competition Between Multiplexed Reactions Quality Control Because cellular expression of 18S rRNA is several magnitudes greater than typical cytokine mRNA expression domination by the 18S reaction is a concern To minimize the competition between the reactions the 18S endogenous control assay is primer limited to prevent it from competing with the amplification of the cytokine target sequences For more information about controlling competition between reactions and the primer limitation concept see the following publications ABI PRISM 7700 Sequence Detection System User Bulletin 2 Relative Quantitation of Gene Expression P N 4303859 ABI PRISM 7700 Sequence Detection System User Bulletin 5 Multiplex PCR with TaqMan VIC Probes P N 4306236 Functional verification of the preloaded probe
84. tokine gene expression values from data in exported results files To calculate relative quantification values from your exported data follow the procedure for multiplex experiments as outlined in the ABI PRISM 7700 Relative Quantification Software User s Manual P N 4309937 This chapter contains information to help guide you through the analysis and to help you interpret the results Where You Are in the Procedure You Are Here Reverse Transcription cDNA generation PCR ABI Prism 7700 Card Run Launch the Relative Configure the preferences Data Analysis Performed using the Sequence Detection Systems Software Create a template file for Interpreting Results Performed using the ABI Prism 7700 Relative Quantification Software Import the SDS results Analyze and interpret the Interpreting Results Performed using the ABI Prism 7700 Relative Quantification Software Quantification Software for a card analysis configure the analysis method and the C limits TaqMan Human Cytokine Card experiments or If a template file exists create a relative quantification document from the template files from the card runs results Interpreting Results 5 1 Calculating Relative Cytokine Gene Expression Rationale References 5 2 Interpreting Results The ABI PRISM 7700 Relative Quantification Software calculates relative cytokine ge
85. uirements any nonspecific amplification is not detected Theory of Operation A 3 About AmpliTag Gold DNA Polymerase AmpliTaq Gold is a thermal stable DNA polymerase The enzyme has a 5 3 nuclease activity but lacks a 3 5 exonuclease activity Innis et al 1988 Holland et a 1991 With AmpliTaq Gold enzyme Hot Start PCR and Time Release PCR can be introduced into existing amplification systems with little or no modification of cycling parameters or reaction conditions These techniques improve amplification of most templates by lowering background and increasing amplification of specific products Multicomponent Analysis Fluorescent Detection Passive Reference A 4 Theory of Operation Multicomponenting is the term used to distinguish the contribution each individual dye makes to the fluorescent spectra Overlapping spectra from the pure dye components generate the composite spectrum This spectrum represents one fluorescent reading from one well Current pure dye menus available for multicomponent analysis are Function Dye Reporters FAM TET VIC Quenchers TAMRA MGB Passive reference ROX The Passive Reference is a dye included in the TaqMan Universal PCR Master Mix that does not participate in the 5 nuclease assay The Passive Reference provides an internal reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluo
86. verse Transcription 2 5 Thermal Cycling To conduct reverse transcription thermal cycling 2 6 Reverse Transcription Step Action 1 Load the reactions into a thermal cycler 2 Program your thermal cycler with the following conditions Reverse St Hexamer Reverse Transcriptase ep Incubationa Transcription Inactivation HOLD HOLD HOLD Temp 25 C 37 C 95 C Time 10 min 60 min 5 min Volume 100 pL a When using random hexamers for first strand cDNA synthesis a primer incubation step 25 C for 10 min is necessary to maximize primer RNA template binding 3 Begin reverse transcription IMPORTANT After thermal cycling store all cDNA samples at 15 to 25 C and proceed to Chapter 3 PCR PCR Overview Amplification of cDNA is the second step in the TagMan Human Cytokine Card two step RT PCR experiment In this step AmpliTaq Gold DNA polymerase amplifies cDNA synthesized from the original total RNA sample Because each cytokine card can evaluate only one cDNA sample you must repeat this step for each sample in the analysis Where You Are in the Procedure ae ABI Prism 7700 Card Run Reverse Transcription a Prepare PCR sample cDNA generation specific reaction mix cDNA sample TaqMan Universal PCR Master Mix 20X 18S Primer and Probe Mix You PCR b Load the PCR sample Are Here ABI Prism 7700 Card run specific reaction mix

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