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1. RT SYBR Green Fluor 24 8 25 ml qPCR Mastermix Catalog no 330513 330511 330519 Number of array 24 x 96 4 x 384 2000 x 25 pl reactions well well reactions 2x SYBR Green Fluor qPCR 24 x 8 x 25 ml Mastermix containing 1 35 ml 1 35 ml HotStart DNA Taq Polymerase PCR Buffer dNTP mix dATP dCTP dGTP dTTP m SYBR Green dye m Fluorescein passive reference dye Suitable for use with the following real time cyclers Bio Rad models iCycler iQ5 MyiQ MyiQ2 RT Predictor PCR Array Handbook 10 2012 7 RT SYBR Green 24 8 25 ml qPCR Mastermix Catalog no 330523 330521 330529 Number of array 24 x 96 4 x 384 2000 x 25 pl reactions well well reactions 2x SYBR Green ROX qPCR 24 x 8x 25 ml Mastermix containing 1 35 ml 1 35 ml HotStart DNA Polymerase PCR Buffer dNTP mix dATP dCTP dGTP dTTP m SYBR Green dye ROX passive reference dye Suitable for use with the following real time cyclers Applied Biosystems models 5700 7000 7300 7500 Standard and Fast 7700 7900HT Standard and Fast 96 well block 384 well block StepOnePlus ViiA 7 Standard and Fast 96 well block 384 well block Eppendorf Mastercycler ep realplex models 2 25 4 45 Stratagene models Mx3000P Mx3005P Mx4000 Takara TP 800 8 RT Predictor PCR Array Handbook 10 2012 RT SYBR Green 24 8 25 ml FAST Mastermix Catalog no 33062
2. RT Predictor PCR Array Format 384 16 x 24 option 384 well plate containing dried assays 8 Optical Adhesive Film 1 per 384 well plate 8 Handbook 1 Suitable for use with the following real time cycler Roche LightCycler 480 384 well block t For a description of the 384 16 x 24 option see page 14 RT Predictor PCR Array Format R Rotor Disc 100 containing dried assays 24 Rotor Disc Heat Sealing Film 1 per Rotor Disc 100 24 Handbook 1 Suitable for use with the following real time cyclers QIAGEN Rotor Gene Rotor Gene 6000 other Rotor Gene cyclers RT First Strand Kit 12 Catalog no 330401 Number of 20 pl reactions 12 Buffer GE 24 ul 5x Buffer BC3 48 Reverse Transcriptase Mix 24 ul Control P2 12 ul RNase Free Water 1 ml 6 RT Predictor PCR Array Handbook 10 2012 RT SYBR Green qPCR 24 8 25 ml Mastermix Catalog no 330503 330501 330509 Number of array 24 x 96 4 x 384 2000 x 25 pl reactions well well reactions 2x SYBR Green qPCR 24 x 8 x 25 ml Mastermix containing 1 35 ml 1 35 ml HotStart DNA Taq Polymerase PCR Buffer dNTP mix dATP dCTP dGTP dTTP m SYBR Green dye Suitable for use with real time cyclers that do not require a reference dye including Bio Rad models CFX96 CFX384 Bio Rad MJ Research models Chromo4 DNA Engine Opticon 2 Roche LightCycler 480 96 well and 384 well
3. The pathway activity score is calculated based on a standard classification model e g Support Vector Machine and Random Forest with 16 biomarker signature genes that is trained on samples with defined manipulation of pathway activity The prediction of pathway activity of each test sample is a collection of pathway activity scores derived from 50 models trained on randomly selected bootstrapped training data sets The details involved in derivation of pathway signature biomarker genes and classification model development is provided on page 44 All 50 pathway activity scores for a test sample are shown as a box and whisker plot The bottom and top of the box are the 25th and 75th percentile the lower and upper quartiles respectively and the band near the middle of the box is the 50th percentile the median The ends of the whisker indicate the minimum and maximum values and outliers are depicted as small circles Figure 7 A positive value indicates that the pathway is activated in an experimental sample versus a control sample whereas a negative value indicates repression of pathway activity The median probability scores of different samples are indicated in the table below the box and whisker plot A statistical P value will be provided for each sample The P value is calculated based on comparison of the experimental sample to untreated samples of signaling pathway or negative control treatment samples of toxic pat
4. 2 suitable for use with real time cyclers that use ROX reference dye including the Rotor Gene Q and Rotor Gene 6000 2 x 1 35 ml Mastermix Related products Larger kit sizes available please inquire RT Predictor PCR Array Handbook 10 2012 47 Product Contents Cat no Human XpressRef 2 tubes each containing 100 human 338112 Universal Total RNA RNA at 1 mg ml RNeasy Mini Kit 50 50 RNeasy Mini Spin Columns 74104 Collection Tubes 1 5 ml and 2 ml RNase free reagents and buffers RNase Free DNase Set For DNase digestion during RNA 79254 50 purification 1500 units RNase free DNase RNase free Buffer RDD and RNase free water for 50 RNA minipreps RNeasy Micro Kit 50 50 RNeasy MinElute Spin Columns 74004 Collection Tubes 1 5 ml and 2 ml RNase free reagents and buffers PAXgene Blood RNA Kit 50 PAXgene Spin Columns 50 762174 50 PAXgene Shredder Spin Columns Processing Tubes RNase Free DNase RNase free reagents and buffers To be used in conjunction with PAXgene Blood RNA Tubes RNeasy Microarray RNeasy Mini Spin Columns Collection 73304 Tissue Mini Kit 50 Tubes QIAzol Lysis Reagent RNase free reagents and buffers QlAamp RNA Blood 50 QlAamp Mini Spin Columns 50 52304 Mini Kit 50 QlAshredder Spin Columns Collection Tubes 1 5 ml and 2 ml RNase free reagents and buffers RT PCR Array Loading 12 x 5 ml capacity irradiation sterilized 338162 Reservoir loading reservoirs Fo
5. HQ o HQ o Wo n n n D m 12 oa 2 n E 05 os Wk 8 os F 06 06 14 06 14 o6 G 07 15 07 15 07 15 07 15 nrc os 16 RC 16 16 PC 16 PC Figure 5 Loading RT Predictor PCR Arrays Formats A C D or F 96 well 4 x 24 option Add 25 ul PCR components mix into wells corresponding to each sample as indicated in the figure Proceed to step 4 Formats E or G 384 well 16 x 24 option Note Each 384 well plate contains 16 replicates of 24 assays that can be used for analysis of 16 samples with reactions for each sample separated from one another by only one row The spacing between the tips of standard multichannel pipettors allows rows or columns to be skipped when adding each sample Be sure to load each sample into the correct set of wells using Figure 6 as a guide Carefully remove the RT Predictor PCR Array from its sealed bag Optional If the PCR components mix is in a tube transfer to a loading reservoir such as the RT PCR Array Loading Reservoir cat no 338162 Add PCR components mix to each well of the RT Predictor PCR Array using a 12 channel pipettor and use Figure 6 as a guide for each sample RT Predictor PCR Array Handbook 10 2012 27 123 4 5 6 7 8 9 10111213 14 15 16 17 18 19
6. Predictor PCR Array Handbook 10 2012 9 Product Use Limitations RT Predictor PCR Arrays the RT First Strand Kit and RT SYBR Green Mastermixes are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our
7. RNase free water 360 ul 140 ul Total volume 800 ul 360 Note This provides an excess volume of 200 ul formats A C D 96 4 x 24 option 120 pl formats E 384 16 x 24 option to allow for pipetting errors Perform pipetting steps as precisely as possible to ensure that each well receives the required volume Note Save the remaining 10 ul cDNA synthesis reaction at 20 C as it may be needed to perform quality control analysis 3 Dispense the PCR components mix into the RT Predictor PCR Array depending on the RT Predictor PCR Array format as described below Note Change pipet tips following each pipetting step to avoid cross contamination between the wells Note If using an instrument to automate this step contact Technical Service for plate specifications Formats A C D or F 96 well 4 x 24 Note Each 96 well plate contains 4 replicates of 24 assays that can be used for analysis of 4 samples Figure 5 Carefully remove the RT Predictor PCR Array from its sealed bag Optional If the PCR components mix is in a tube transfer to a loading reservoir such as the RT PCR Array Loading Reservoir cat no 338162 Add 25 ul PCR components mix to each well of the RT Predictor PCR Array using an 8 channel pipettor or a 12 channel pipettor using only 8 tips 26 RT Predictor PCR Array Handbook 10 2012 123 45 67 8 9 101112 ow o o
8. data analysis method Due to the inverse proportional relationship between the threshold cycle C and the original gene expression level and the doubling of the amount of product with every cycle the original expression level L for each gene of interest is expressed as L 2 67 To normalize the expression level of a gene of interest to housekeeping gene HKG the expression levels of the two genes are divided 2 Ci GOI 2 Cr HKG 2 Cr GOI Cr HKG 2 To determine fold change in gene expression the normalized expression of the GOI in the experimental sample is divided by the normalized expression of the same GOI in the control sample 2 1 lt 9 AAC A i 2 AC conirol 2 T Where AAC is equal to ACr expt 44 RT Predictor PCR Array Handbook 10 2012 The complete calculation is as follows 2 2 2 1 C H 274C expt _________________ LL 444 2 C GO control 9 IC GOI E control 274 control 2 control RT Predictor PCR Array Handbook 10 2012 45 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references
9. realplex models 2 25 4 45 Stratagene models Mx3005P Mx3000P Takara TP 800 RT Predictor PCR Array Format 96 well plate containing dried assays 24 Optical Adhesive Film 1 per 96 well plate 24 Handbook 1 t Suitable for use with the following real time cyclers Applied Biosystems models 7500 Fast block 7900HT Fast block StepOnePlus ViiA 7 Fast block RT Predictor PCR Array Format D 96 well plate containing dried assays 24 Optical Thin Wall 8 Cap Strips 12 per 96 well plate 288 Handbook 1 Suitable for use with the following real time cyclers Bio Rad CFX96 Bio Rad MJ Research models DNA Engine Opticon DNA Engine Opticon 2 Stratagene Mx4000 RT Predictor PCR Array Format E 384 16 x 24 option 384 well plate containing dried assays 8 Optical Adhesive Film 1 per 384 well plate 8 Handbook 1 Suitable for use with the following real time cyclers Applied Biosystems models 7900 384 well block ViiA 7 384 well block Bio Rad CFX384 t For a description of the 384 16 x 24 option see page 14 ct a H M HH M g RT Predictor PCR Array Handbook 10 2012 5 RT Predictor PCR Array Format 96 well plate containing dried assays 24 Optical Adhesive Film 1 per 96 well plate 24 Handbook 1 1 Suitable for use with the following real time cycler Roche LightCycler 480 96 well block
10. Green Mastermixes uniquely provide more accurate SYBR Green results by preventing the amplification of primer dimers and other nonspecific products They also help ensure high amplification efficiencies even for genes that are difficult to amplify Real time cyclers use different reference dyes to normalize their optics therefore be sure to use the correct mastermix for the real time cycler in your laboratory The RT First Strand Kit includes a proprietary buffer to eliminate any residual genomic DNA contamination in RNA samples before it can be amplified into secondary products that would otherwise cause false positive signals The reverse transcription controls RTC on the RT Predictor PCR Array can only be r aaaea aaa 12 RT Predictor PCR Array Handbook 10 2012 evaluated with the built in external RNA control of the RT First Strand Kit These controls do not yield results when used with other sources of reverse transcriptase or first strand synthesis kits The buffer components and the magnesium concentration in the RT First Strand Kit are also more compatible with RT SYBR Green Mastermixes than other enzymes or kits providing the RT Predictor PCR Arrays with maximum levels of sensitivity with nanogram to microgram amounts of total RNA Principle and procedure RT Predictor PCR Arrays are provided in 96 well plates 384 well plates or Rotor Discs Figures 1 3 RT Predictor PCR Arrays in 96 well plates contain
11. PPC consists of a predispensed artificial DNA sequence and the assay that detects it This control tests the efficiency of the polymerase chain reaction itself Controls can be used to test for inter sample intra plate consistency RT Predictor PCR Array Handbook 10 2012 13 a e 1209000002 27000000 3 ex mc 1 2 Aw ow 12 F 06 14 Gw s H 16 Figure 1 RT Predictor PCR Array Formats A C D F 4 x 24 layout RT Predictor PCR Array Formats A C D and F Each plate contains 4 sets of real time PCR assays for pathway activity assessment Each set of genes contains 16 pathway specific signature genes 1 16 5 housekeeping genes HK1 5 to normalize data one genomic DNA control GDC one reverse transcription control RTC and one positive PCR control PPC 12345267 8 910111213 1415 16 17 18 19 202122 23 24 A 62 69 64 65 66 27 v 2 15 49 1 e 04 05 06 07 0 72 13 ac de C or 64 65 66 09 BAGH wo D Gi e 4 05 07 D wc E 01 mo 0 07 0 0 xc F er 2 3 0 05 G6 Gr 09 09 0 GD 12 13 GH mo G or o2 o4 65 o6 o7 o8 0 GH 1213 o er e 04 05 66 7 09 10 GD 12 15 TH 01 02 05 06 07 0 0
12. errors Perform pipetting steps as precisely as possible to ensure that each well receives the correct volume 32 RT Predictor PCR Array Handbook 10 2012 Note Save the remaining 10 ul cDNA synthesis reaction at 20 C as it may be needed to perform quality control analysis using the RT RNA QC PCR Array Carefully remove the RT Predictor PCR Array from its sealed bag Slide the array into the Rotor Disc 100 Loading Block using the tab at position A1 and the tube guide holes Add 20 pl PCR components mix to each well of the RT Predictor PCR Array Proceed to step 5 Note Change pipet tips following each pipetting step to avoid cross contamination between the wells Note PCR components mix can be dispensed manually or using the QlAgility www giagen com goto GlAgility Note Although wells 97 100 do not contain assays it is essential to add PCR components mix for optimized balancing of the RT Predictor PCR Array Carefully seal the RT Predictor PCR Array with Rotor Disc Heat Sealing Film using the Rotor Disc Heat Sealer For detailed instructions see the Rotor Gene Q User Manual Note The RT Predictor PCR Array containing PCR components mix may be stored at 20 C wrapped in aluminum foil for up to one week if desired Program the real time cycler according to Table 8 Note For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www SABiosciences com pcrarray
13. handbook contains 4 protocols The first protocol details cDNA synthesis by reverse transcription using purified RNA and the RT First Strand Kit page 23 This protocol should be performed prior to real time PCR The 3 additional protocols detail real time PCR performed using the cDNA prepared in the first protocol as the template The protocol on page 25 should be used for 96 well and 384 well RT Predictor PCR Arrays Formats A C D E F and G The protocol on page 32 should be used for Rotor Disc 100 RT Predictor PCR Array Format R 16 RT Predictor PCR Array Handbook 10 2012 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier In addition to the RT Predictor PCR Array RT First Strand Kit and RT SYBR Green Mastermix the following are required E Purified RNA samples Real time PCR cycler High quality nuclease free water Do not use DEPC treated water Multichannel pipettor Single channel pipettor Nuclease free pipet tips and tubes Optional XpressRef Universal Total RNA to control PCR conditions is available for human cat no 338112 ee essi RT Predictor PCR Array Handbook 10 2012 17 Important Notes Preparing a workspace free of DNA contamination For accurate and reproducible RT Predicto
14. invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding RT Predictor PCR Arrays the RT First Strand Kit RT SYBR Green Mastermixes or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN 10 RT Predictor PCR Array Handbook 10 2012 Technical Service Departments or local distributors see back cover or visit www qiagen com Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please cons
15. iterations the median rank of each gene was calculated and the top 16 genes were selected as the final signature biomarker gene set for assessing specific pathway activity A new set of 50 bootstrapped samples were drawn and used to train the final model ensemble with the selected final 16 biomarker signature genes and the resulting 50 models are used to predict the pathway activity score of test samples All 50 predictions for a test sample are shown as a box and whisker plot Figure 7 Principle of calculation between control and experimental groups for 96 well and 384 well formats Data analysis for formats A C D E F and is described A1 Change all values reported as greater than 35 or as N A not detected to 35 At this point any value equal to 35 is considered a negative call A2 Examine the values of the genomic DNA control wells GDC as follows Calculate 1995 If the value is greater than 35 level of genomic DNA contamination is too low to affect gene expression profiling results No action is needed If the value is less than 35 genomic DNA contamination is evident See the Troubleshooting Guide page 36 A3 Examine the value of the reverse transcription control RTC of each sample using the value for the corresponding positive PCR control PPC as follows B Calculate AC m If this value is less than 5 then no inhibition of the reverse tra
16. keeping genes can be excluded from analysis of the average of HKG C values in the equation above If an appropriate housekeeping gene has not been previously identified use the average C value of all housekeeping genes A6 When biological and or technical replicates are performed calculate the average AC value of each gene each well across those replicate arrays for each treatment group A7 Calculate the AAC for each gene across 2 samples of RT Predictor PCR Arrays or groups of samples Use the formula AAC AC group 2 AC group 1 where group 1 is the control sample or group of control samples and group 2 is the experimental sample or group of experimental samples A8 Calculate the fold change for each gene from group 1 to group 2 as 2 AACT Note If the fold change is greater than 1 the result may be reported as a fold upregulation If the fold change is less than 1 the negative inverse of the result may be reported as a fold downregulation Fold change ratio calculation will not be reliable when raw C values from both groups are greater than 35 Note The free online software GNCPro outlines gene and pathway interactions http geneweb SABiosciences com 42 RT Predictor PCR Array Handbook 10 2012 Principle of AAC calculation between control and experimental groups for Rotor Disc formats A1 Change all C values reported as greater than 33 or as N A not detected to 33 At this point any valu
17. php 28 RT Predictor PCR Array Handbook 10 2012 Table 4 Cycling conditions for Applied Biosystems Bio Rad Stratagene and Eppendorf cyclers Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Polymerase 15 activated by this heating step 40 15 5 95 1 min 60 Perform fluorescence dota collection Recommended for the following cyclers Applied Biosystems models 5700 7000 7300 7500 7700 7900HT StepOnePlus ViiA 7 Bio Rad models iCycler iQ5 MyiQ MyiQ2 CFX96 CFX384 Stratagene models Mx3000P Mx3005P Mx4000P Eppendorf Mastercycler ep realplex models 2 25 4 45 t For Bio Rad models CFX96 and CFX384 adjust the ramp rate to 1 C s For Eppendorf Mastercyler ep realplex models 2 2S 4 and 4S for the Silver Thermoblock adjust the ramp rate to 2696 for the Aluminum Thermoblock adjust the ramp rate to 35 Refer to the Instrument Setup Guide at www SABiosciences com pcrarrayprotocolfiles php for detailed setup instructions Table 5 Cycling conditions for Roche LightCycler 480 Cycles Duration Temperature Comments 1 10 min 95 HotStart Polymerase is activated by this heating step 45 15s 25 C 1 min 60 C Perform fluorescence data collection Recommended for the Roche LightCycler 480 If using a Roche LightCycler 480 adjust the ramp rate to 1 C s Refer to the Instrument Setup Guide at www SABiosciences com pcrarrayproto
18. this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product of its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 The purchase of this product includes a limited non transferable license under specific claims of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology Inc and Roche Diagnostics GmbH to use only the enclosed amount of product according to the specified protocols No right is conveyed expressly by implication or by estoppel to use any instrument or system under any claim of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 oth
19. visit the reference database online at www sABiosciences com support_publication php pcrarray or contact QIAGEN Technical Services or your local distributor Eram 46 RT Predictor PCR Array Handbook 10 2012 Ordering Information Product Contents Cat no RT Predictor PCR Array Arrays of assays for pathway activity or 330232 biological function available in 96 well 384 well and Rotor Disc 100 formats RT RNA QC PCR Array Array for quality control analysis prior 330291 to experiments using RT Predictor PCR Arrays available in 96 well 384 well and Rotor Disc 100 formats RT First Strand Kit 12 For 12 x 20 pl first strand cDNA 330401 synthesis reactions Buffer GE 30 ul Buffer BC3 60 ul Reverse Transcriptase Mix 28 pl Control P2 18 ul RNase Free Water 1 ml RT SYBR Green qPCR For 2 x 96 assays in 96 well plates 330500 Mastermix 2 suitable for use with real time cyclers that do not require a reference dye 2x 1 35 ml Mastermix RT SYBR Green Fluor For 2 x 96 assays in 96 well plates 330510 qPCR Mastermix 2 suitable for use with real time cyclers that use fluorescein reference dye 2x 1 35 ml Mastermix RT SYBR Green ROX For 2 x 96 assays in 96 well plates 330520 qPCR Mastermix 2 suitable for use with real time cyclers that use ROX reference dye 2 x 1 35 ml Mastermix RT SYBR Green ROX For 2 x 96 assays in 96 well plates 330620 FAST Mastermix
20. 13 Recommended Perform dissociation melting curve analysis to verify PCR specificity Run a melting curve program and generate a first derivative dissociation curve for each well using the real time cycler software A single peak should appear in each reaction at temperatures greater than 80 C Note If your instrument does not have a default melting curve program run the following program instead 95 C 1 min 65 C 2 min optics off 65 C to 95 C at 2 C min optics Note For cycler specific melting curve analysis settings please refer to the Instrument Setup Guide for your cycler at www SABiosciences com pcrarrayprotocolfiles php Note Plates can be stored at 20 C wrapped in aluminum foil and melting curve analysis performed at a later time When ready to perform melting curve analysis warm the plate to room temperature 15 25 place it in the real time cycler and run the melting curve analysis program Note Visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed note which wells are affected as this may affect the results of data analysis Note Do not open any previously processed RT Predictor PCR Array Removing the Optical Thin Wall 8 Cap Strips or the Optical Adhesive Film from RT Predictor PCR Arrays releases PCR product into the air where it may contaminate and affect the results of future real time PCR experiments RT Predictor PCR Arra
21. 2 13 14 15 16 1 HK2 HKS GDC RIC 16 Figure 6 Loading RT Predictor PCR Arrays Formats E or G 384 well 16 x 24 option Add 10 ul PCR components mix into wells corresponding to each sample as indicated in the figure Proceed to step 4 4 Carefully tightly seal the RT Predictor PCR Array with Optical Thin Wall 8 Cap Strips Formats A and D or Optical Adhesive Film Formats C E F and G IMPORTANT Users of Bio Rad and Eppendorf real time cyclers must ensure that the real time cycler has been calibrated to use clear flat optical caps with RT Predictor PCR Array plates prior to initiating the run 5 Centrifuge for 1 min at 1000 g at room temperature 15 25 to remove bubbles Visually inspect the plate from underneath to ensure no bubbles are present in the wells Note The presence of bubbles in the wells interferes with results 6 Place the RT Predictor PCR Array on ice while setting up the PCR cycling program Note The RT Predictor PCR Array containing PCR components mix may be stored at 20 C wrapped in aluminum foil for up to one week if desired 7 Program the real time cycler according to Table 4 5 or 6 depending on the real time cycler used If prompted by your cycler software select Absolute Quantitation to begin Note For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www SABiosciences com pcrarrayprotocolfiles
22. 2021 22 23 24 Samples A 01 02 04 05 06 07 08 09 10 11 12 13 14 15 16 HK3 KS GDC RIC 1 B 02 04 05 06 07 08 09 10 11 12 13 14 15 16 HK2 HK4 AKS GDC RIC 2 C 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HK2 5 GDC RTC 3 D 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HK HK2 5 GDC RTC 4 E 01 02 03 04 05 06 07 09 10 11 12 13 14 15 16 AKI HK2 HKS GDC RTC 5 F 1 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HKI HK2 5 GDC RIC PPC 6 G 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 K HKS GDC RIC 7 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HK2 HK3 5 GDC RTC 8 01 02 04 05 06 07 08 09 10 11 12 13 14 15 16 HK2 HK4 AKS GDC RTC 9 J 02 04 05 06 07 08 09 10 11 12 13 14 15 16 HK HK3 AKA HKS GDC RIC 10 K 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HKD HK2 5 GDC RTC n L 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 K HK2 HK3 5 GDC RTC 12 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HKD HK2 HK5 GDC RIC 13 N 02 04 05 06 07 08 09 10 11 12 13 14 15 16 KA HKS GDC RIC 14 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 HKD HK2 HK5 GDC RTC 15 P 02 04 05 06 07 08 09 10 11 1
23. 3 330621 330629 Number of array 24x96 4 384 2000 25 pl reactions well reactions 2x SYBR Green ROX FAST 24 x 8x 25 ml Mastermix containing 1 35 ml 1 35 ml HotStart DNA Polymerase m PCR Buffer dNTP mix dATP dCTP dGTP dTTP m SYBR Green dye m ROX passive reference dye Suitable for use with the Rotor Gene Q QIAGEN and Rotor Gene 6000 Note RT Predictor PCR Arrays cannot be used in the Cepheid SmartCycler or the Roche LightCycler 2 0 Shipping and Storage RT Predictor PCR Array Formats A C D E F G and R are shipped at room temperature 15 25 or on ice or dry ice depending on the destination and accompanying products All RT Predictor PCR Array Formats should be stored at 20 C upon arrival When stored properly at 20 C RT Predictor PCR Arrays are stable for up to 6 months after delivery RT SYBR Green Mastermixes are shipped on cold packs For long term storage keep tubes at 20 C If the entire volume will not be used at once we recommend dividing into aliquots and storing at 20 Avoid repeated freezing and thawing If stored under these conditions RT SYBR Green Mastermixes are stable for 6 months after receipt The RT First Strand Kit is shipped frozen For long term storage keep the kit at 20 C If stored under these conditions the RT First Strand Kit is stable for 6 months after receipt We recommend a maximum of 6 freeze thaw cycles RT
24. 7 2 134 xe J 01 02 08 04 05 06 07 08 op wo er 0 04 65 06 67 08 09 09 0 02 09 14 ac L 62 6 0 65 47 12 15 6 ac de M er es 4 09 6 o7 08 09 00 0 02 09 GH ac No mm 04 o 0 w o xc 2 05 06 mc de P e 62 65 6 05 06 07 09 0 1 12 4 E Figure 2 RT Predictor PCR Array Formats E and 384 16 x 24 option layout RT Predictor PCR Arrays with the 384 16 x 24 option include 16 replicates of the same 24 assays that are provided in 4 replicates in the 96 well format shown in Figure 1 14 RT Predictor PCR Array Handbook 10 2012 097 Sa e e co e O ee gt e d e a 9 er 2 e e e amp E amp amp e amp e o amp amp e o amp e o9 e 2 e E e e e fs 20 Se oe Seen Figure 3 RT Predictor PCR Array Format R layout RT Predictor PCR Array Format R contains the same 4 replicates of 24 assays wells 1 24 25 48 49 72 and 73 96 as in the 96 well format shown in Figure 1 Each set of genes contains 16 pathway specific signature genes 5 housekeeping genes HK to normalize data one genomic DNA control GDC one reverse transcription control RTC and one positive PCR control PPC Wells 97 to 100 are emply Workflow The procedure begins with the conversion of experimental RNA samples in
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26. Array to the following terms 1 The RT Predictor PCR Array may be used solely in accordance with the RT Predictor PCR Array Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the RT Predictor PCR Array Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www giagen com LIMITED LICENSE STATEMENTS Use of this product is covered by one or more of the following US patents and correspon
27. October 2012 RT Predictor PCR Array Handbook RT Predictor PCR Array RT First Strand Kit RT SYBR Green qPCR Mastermix RT SYBR Green Fluor qPCR Mastermix RT SYBR Green qPCR Mastermix RT SYBR Green ROX FAST Mastermix For biological function identification using real time RT PCR analysis QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 5 Shipping and Storage 9 Product Use Limitations 10 Product Warranty and Satisfaction Guarantee 10 Technical Assistance 10 Safety Information 11 Quality Control 11 Introduction 12 First strand cDNA synthesis and mastermixes 12 Principle and procedure 13 RNA quality control using an RT RNA QC PCR Array 16 Description of protocols 16 Equipment and Reagents to Be Supplied by User 17 Important Notes 18 Preparing a workspace free of DNA contamination 18 RNA preparation quantification and q
28. RT Predictor PCR Array format are suitable for your real time cycler see page 5 9 The format of the RT Predictor PCR Array is indicated by the last letter of the catalog number An incorrect RT Predictor PCR Array format will not fit the real time cycler properly and may damage the real time cycler Do notcut the plastic plate of the RT Predictor PCR Array For accuracy and precision ensure that micropipettors are calibrated before beginning the protocol Be sure not to introduce bubbles into the wells of the RT Predictor PCR Array when pipetting Do not use DEPC treated water Use high quality nuclease free water M If precipitates are present in the Mastermix tubes warm the reagents at 42 for 1 min and vortex briefly to dissolve Repeat if necessary Procedure 1 Briefly centrifuge the RT SYBR Green Mastermix 10 15 s to bring the contents to the bottom of the tube Note As the RT SYBR Green Mastermix contains HotStart DNA Taq Polymerase that is active only after heat activation reactions can be prepared at room temperature 15 25 C 2 Prepare the PCR components mix in a 1 5 ml tube or a loading reservoir depending on the RT Predictor PCR Array format as described in Table 3 RT Predictor PCR Array Handbook 10 2012 25 Table 3 PCR components mix 96 well 4 x 24 384 16 x 24 option Array format A C D F E 2x RT SYBR Green 400 ul 180 ul Mastermix cDNA synthesis reaction 40 ul 40 ul
29. a spectrophotometer Prepare dilutions and measure absorbance in 10 mM Tris Cl pH 8 0 The spectral properties of nucleic acids are highly dependent on pH An absorbance reading of 1 0 at 260 nm in a 1 cm detection path corresponds to an RNA concentration of 40 ug ml ratio should be greater than 1 7 ratio should be 1 8 to 2 0 Concentration determined by should be gt 40 ug ml Ribosomal RNA band integrity Run an aliquot of each RNA sample on a denaturing agarose gel or the Agilent Bioanalyzer using RNA 6000 Nano LabChip Verify that there are sharp bands peaks present for both the 18S and 28S ribosomal RNAs Figure 5 Any smearing of the RNA bands or shoulders on the RNA peaks indicates that degradation has occurred in the RNA sample For reliable data from RT Predictor PCR Arrays an RNA Integrity Number RIN of 7 or higher is recommended Consistent RIN values across multiple samples within each experiment are desirable for reliable quality data comparisons When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs available from the product supplier 20 RT Predictor PCR Array Handbook 10 2012 FU MDA231 lt 285 lt 185 i NNW 8 T in 1 1 20 25 30 35 40 45 50 55 5 Figure 5 Ribosomal RNA in
30. athway signature biomarker gene selection and model derivation A classification algorithm such as Random Forest was trained on the training sample set using experimentally selected 100 200 genes for each pathway n order to evaluate classifier model performance without selection bias the bootstrap resampling method was used to randomly split training samples set into two parts training and verification subsets in an 8596 to 1596 ratio The bootstrap was carried out without replacement and stratified by class pathway activated and pathway repressed samples were selected proportionally The top 16 genes were selected based on ranked gene list according to the classification model variable importance measure the mean decrease in out of bag classifier accuracy when a gene s expression value is randomly permuted Anew Random Forest or Support Vector Machine classifier model was trained by using only the top 16 genes This model was used to score the 15 out of training verification samples to estimate the classifier performance 40 RT Predictor PCR Array Handbook 10 2012 About 250 bootstrap resamplings were iterated and each resampling generated a slightly different set of top 16 genes The classification accuracy specificity and other characteristics were estimated using the out of bootstrap sample activity scores from the 250 iterations with their corresponding top 16 genes and classifier models After 250
31. colfiles php for more information on other required changes to settings for Melt Curve Acquisition RT Predictor PCR Array Handbook 10 2012 29 Table 6 Cycling conditions for Bio Rad and Takara cyclers and all other cyclers Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Polymerase 15 activated by this heating step 40 15 5 95 30 40 s 5536 Perform fluorescence data collection Different cyclers need different lengths of time to detect the fluorescent signal Choose the appropriate time for the annealing step 55 C for your cycler 30 s 725C t Recommended for the following cyclers Bio Rad MJ Research models Chromo4 DNA Engine Opticon DNA Engine Opticon 2 Takara TP 800 all other cyclers 8 Place the RT Predictor PCR Array in the real time cycler If recommended by the cycler user manual use a compression pad with RT Predictor PCR Arrays sealed with optical adhesive film formats C E F and G Start the run 9 Calculate the threshold cycle C for each well using the real time cycler software as described in the following steps Note If using the Roche LightCycler 480 there are 2 options for data analysis using the second derivate max setting in this case there is no need to calculate the or using Fit Points in this case the should be defined manually as described in step 11 10 Define the baseline by choosing the automated baseline option if the cyc
32. ding patent claims outside the US 5 994 056 and 6 171 785 The purchase of this product includes a limited nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA This product is provided under an agreement between Molecular Probes Inc and QIAGEN and the manufacture use sale or import of this product is subject to one or more of U S Patent Nos 5 436 134 5 658 751 and corresponding international equivalents owned by Invitrogen Corp The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer where such research does not include testing analysis or screening services for any third party in return for compensation on a per test basis The buyer cannot sell or otherwise transfer a
33. e RNeasy Mini Kit b RT First Strand Kit not We strongly recommend using the RT First used Strand Kit for cDNA synthesis This kit includes a genomic DNA elimination step c Reagents tips or tubes See Preparing a workspace free of DNA contaminated contamination page 18 If using the RT RNA QC PCR Array the no template control NTC indicates the level of DNA contamination in the experimental setup d Genomic DNA difficult Pathway activity change calls may still be to remove obtained However it is very important to verify any results with further samples that have undergone more rigorous steps to eliminate genomic DNA Inefficient reverse transcription Poor quality RNA Check the and A 555 473o ratios of RNA samples Be sure to perform the dilutions for spectrophotometry in RNase free Tris pH 8 0 If necessary repurify RNA using a spin column method such as the RNeasy Mini Kit 36 RT Predictor PCR Array Handbook 10 2012 Comments and suggestions Poor PCR amplification efficiency a Real time cycler Real time cyclers vary in their level of sensitivity sensitivity If a C value of 20 2 is difficult to obtain from the positive PCR control PPC the observed CPS value should be acceptable as long as it does not vary by more than 2 cycles between RT Predictor PCR Arrays b Cycling program Be sure that the initial heat activation step at incorrect 95 C was lengthened to 10 minutes from the shorter t
34. e equal to 33 is considered a negative call A2 Examine the C values of the genomic DNA control wells GDC as follows Calculate 1995 If the value is greater than 33 level of genomic DNA contamination is too low to affect gene expression profiling results No action is needed m If the value is less than 33 genomic DNA contamination is evident See the Troubleshooting Guide page 36 A3 Examine the values of the reverse transcription control RTC using the values for the positive PCR control PPC as follows B Calculate AC m If this value is less than 5 then no inhibition of the reverse transcription reaction is apparent No action is needed If this value is greater than 5 there is evidence of impurities that may have inhibited the reverse transcription reaction See the Troubleshooting Guide page 36 A4 Examine the C values of the positive PCR control wells PPC as follows Calculate The CS value should be 14 2 of each sample on the RT Predictor PCR Array and should not vary by more than 2 cycles between different samples of RT Predictor PCR Arrays being compared m Larger differences values between samples indicate the presence of PCR amplification inhibitors This means that the RNA samples require further purification value of that is consistently greater than 16 for all samples may indicate a problem with the cycling c
35. e same amount of total RNA for reverse transcription of each sample First time users are recommended to start with 300 ng total RNA for each sample Use of less than 100 ng RNA will result in a high rate of false negatives that will affect the pathway activity regulation readout Do not use DEPC treated water Use high quality nuclease free water E The RT First Stand Kit is not compatible with the chemicals in DNA free kits from Ambion If your RNA sample has been treated with DNA free reagents contact QIAGEN Technical Service Procedure 1 Thaw the reagents of the RT First Strand Kit Briefly centrifuge 10 15 s to bring the contents to the bottom of the tubes 2 Prepare the genomic DNA elimination mix for each RNA sample according to Table 1 Mix gently by pipetting up and down and then centrifuge briefly Table 1 Genomic DNA elimination mix Component Amount RNA 100 ng 1 ug Buffer GE 2 pl RNase free water Variable Total volume 10 pl f using the kit for the first time use the RNA amount recommended in Important points before starting above 3 Incubate the genomic DNA elimination mix for 5 min at 42 C then place immediately on ice for at least 1 min RT Predictor PCR Array Handbook 10 2012 23 4 Prepare the reverse transcription mix according to Table 2 Table 2 Reverse transcription mix Component Volume Volume Volume for 1 for 2 for 4 reaction reactions reactio
36. er genes defined as a set of experimentally derived genes in the array whose combined relative expression delivers optimal prediction power for signaling or toxic pathway activity Multiple sets of signature genes in an RT Predictor PCR Array plate allow for testing pathway activity regulation from multiple samples on a single plate for faster throughput RT Predictor PCR Arrays RT SYBR Green Mastermixes and the RT First Strand Kit have been optimized in combination for SYBR Green based real time RT PCR detection providing the RT Predictor PCR Arrays with superior sensitivity and reliable measurement of biological function The simplicity of the RT Predictor PCR Arrays makes them accessible for routine use in every research laboratory First strand cDNA synthesis and mastermixes Performance of RT Predictor PCR Arrays is only guaranteed when used with RT SYBR Green Mastermixes and the RT First Strand Kit The combination of RT Predictor PCR Arrays with RT SYBR Green Mastermixes and the RT First Strand Kit ensures that the PCR assay performance of each signature gene is comparable to what was used to derive the algorithm for biological function prediction Therefore the use of the complete RT Predictor PCR Array System is absolutely essential for obtaining accurate measurement of biological function The chemically modified and tightly controlled HotStart DNA Taq Polymerase enzyme and other proprietary chemical components in RT SYBR
37. er than for the amount of product contained herein The purchase of this product products comprising ROX dye includes a limited non transferable right to use the purchased amount of the product to perform Applied Biosystems patented Passive Reference Method for the purchaser s own internal research No right under any other patent claim and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 2012 QIAGEN all rights reserved _ __ _3_ _ _ www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885
38. hway in training data set Samples with P 0 05 as well as pathway activity score 70 3 or 0 3 indicate a significant change of pathway activity as compared to control Figure 7B RT Predictor PCR Array Handbook 10 2012 39 Classification model and signature biomarker genes derivation from training data set A training sample set and the pathway biomarker genes selection algorithm such as Random Forest was used to select a subset of genes which are able to predict the intracellular status of pathway activity Training sample sets Signaling pathway RT Predictor PCR Arrays The training set contains 15 to 20 different cell line samples with specific alteration to the signaling pathway under evaluation The change in signaling pathway activity upregulation as well as downregulation was experimentally confirmed Toxicity pathway RT Predictor PCR Arrays The training set contains 20 40 HepG2 samples uniquely treated by a number of chemicals which were experimentally verified to manipulate the toxic pathway under evaluation A genomewide microarray experiment was carried out on 1 3 training samples and a 100 200 gene list was selected based on statistical significance of gene expression changes after defined signaling or toxic pathway activity manipulation The expression levels of selected 100 200 genes were analyzed using PCR on the whole training sample set and were used for pathway activity biomarker gene selection P
39. ime in the default program Be sure that all other cycle parameters also have been correctly entered according to the recommendations in the protocol c Poor quality RNA Check the and Ax60 Ao39 ratios of RNA samples Be sure to perform the dilutions for spectrophotometry in RNase free Tris pH 8 0 If necessary repurify RNA using a spin column method such as the RNeasy Mini Kit ee i RT Predictor PCR Array Handbook 10 2012 37 Appendix A Data Analysis Visit our free PCR Predictor Array Data Analysis Web Portal at http pcrdataanalysis sabiosciences com predictorpcrarray At the PCR Predictor Array Data Analysis Web Portal C data can be entered and the web based software will automatically calculate a pathway activity probability score based on the AAC values between control and experimental groups The PCR Array Data Analysis Web Portal presents the pathway activity probability score in a box and whisker plot a table of median probability score and a statistical P value for each group Derivation of pathway activity probability score based on signature biomarker gene expression between experimental and control samples The pathway activity probability score is shown as a box and whisker plot and indicates whether the pathway is activated repressed or whether activity remains unchanged in an experimental sample compared to a control sample Figure 7A 1 0 0 8 0 6 0 4 0 2 00 02 04 06 08 1 0 Probability
40. ler has the adaptive baseline function If the cycler does not have the adaptive baseline function set the baseline manually To set the baseline manually use the linear view of the amplification plots to determine the earliest visible amplification Set the cycler to use the readings from cycle number 2 through 2 cycles before the earliest visible amplification but no more than cycle 15 The earliest amplification will usually be visible between cycles 14 and 18 11 Manually define the threshold by using the log view of the amplification plots Choose a threshold value above the background signal but within the lower one third to lower one half of the linear phase of the amplification plot Note Ensure that the threshold values are the same across all RT Predictor PCR Array runs in the same analysis The absolute position of the threshold 30 RT Predictor PCR Array Handbook 10 2012 is less critical than its consistent position across arrays If the RNA sample is of sufficient quality the cycling program has been carried out correctly and threshold values have been defined correctly the value of should be 20 2 for all arrays or samples 12 Export the values for all wells to a blank Excel spreadsheet for use with the SABiosciences PCR Predictor Array Data Analysis Web based software Note Web based PCR Array Data Analysis Software is available at http pcrdataanalysis sabiosciences com predictorpcrarray
41. ng curve analysis performed at a later time When ready to perform melting curve analysis warm the plate to room temperature 15 25 C place it in the real time cycler and run the melting curve analysis program RT Predictor PCR Array Handbook 10 2012 Note Visually inspect the Rotor Disc after the run for any signs of evaporation from any of the wells If evaporation is observed note which wells are affected as this may affect the results of data analysis Note Do not open any previously processed RT Predictor PCR Array Removing the film from RT Predictor PCR Arrays releases PCR product into the air where it may contaminate and affect the results of future real time PCR experiments RT Predictor PCR Array Handbook 10 2012 35 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www SABiosciences com support fag php target PCR The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Presence of genomic DNA contamination a DNase digestion not We strongly recommend performing the on performed column DNase digestion step when purifying RNA using th
42. ng the log view of the amplification plots Choose a threshold value above the background signal The threshold value should be in the lower half of the linear phase of the amplification plot Note Ensure that the threshold values are the same across all RT Predictor PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays If the RNA sample is of sufficient quality the cycling program has been carried out correctly and threshold values have been defined correctly the value of should be 14 2 for all arrays or samples Export the C values for all wells to a blank Excel spreadsheet for use with the PCR Predictor Array Data Analysis Web based software Note Web based Predictor PCR Array Data Analysis Software is available at http pcrdataanalysis sabiosciences com predictorpcrarray Recommended Perform dissociation melting curve analysis to verify PCR specificity Run a melting curve program and generate a first derivative dissociation curve for each well using the real time cycler software A single peak should appear in each reaction Note Melting curve analysis can be added during creation of the Rotor Gene Q PCR program Note For Rotor Gene Q melting curve analysis settings refer to the Instrument Setup Guide at www SABiosciences com pcrarrayprotocolfiles php Note Rotor Discs can be stored at 20 C wrapped in aluminum foil and melti
43. ng the optional on column DNase digestion step followed by cDNA synthesis using the RT First Strand Kit If required individual species specific RT qPCR Primer gDNA Controls are available RT Predictor PCR Array Handbook 10 2012 21 Starting RNA amounts The RT Predictor PCR Array System provides results with as little as 100 ng or as much as 1 ug total RNA per sample The optimal amount of starting material depends on the relative abundance of the transcripts of interest Lower abundance transcripts require more RNA higher abundance transcripts require less RNA Greater amounts of input total RNA yield a greater number of positive calls i e genes expressed in the linear dynamic range of the method Lower amounts of input total RNA yield a smaller number of positive calls and an increase in false negative calls For successful results and maximum positive call rates that are important for determination of pathway activity regulation we recommend that first time users start with 300 ng total RNA for each sample It is important to use a consistent amount of total RNA for all samples in a single experiment 22 RT Predictor PCR Array Handbook 10 2012 Protocol cDNA Synthesis Using the RT First Strand Kit Use of the RT First Strand Kit is critical for obtaining optimal results and for detection of the reverse transcription controls contained in the RT Predictor PCR Array Important points before starting Use th
44. ns 5x Buffer BC3 4 ul 8 ul 16 ul Control P2 1 ul 2 ul 4 ul RE3 Reverse Transcriptase Mix 2 ul 4 ul 8 ul RNase free water 3 ul 6 ul 12 ul Total volume 10 pl 20 pl 40 5 Add 10 ul reverse transcription mix to each tube containing 10 pl genomic DNA elimination mix Mix gently by pipetting up and down 6 Incubate at 42 for exactly 15 min Then immediately stop the reaction by incubating at 95 C for 5 min 7 Add 30 pl RNase free water to each reaction Mix by pipetting up and down several times 8 Place the reactions on ice and proceed with the real time PCR protocol If you wish to store the reactions prior to real time PCR transfer them to a 20 freezer For quality control analysis using the RT RNA QC PCR Array follow the protocol in the RT RNA QC PCR Array Handbook using a 6 ul aliquot of the diluted cDNA template 24 RT Predictor PCR Array Handbook 10 2012 Protocol Real Time PCR for RT Predictor PCR Arrays Formats A C DE F G This protocol describes real time PCR using RT Predictor PCR Arrays in combination with RT SYBR Green Mastermixes Use of RT SYBR Green Mastermixes is critical to obtain optimal results from the RT Predictor PCR Array If unsure of the RNA quality or purification technique examine the RNA quality before performing this protocol using species and cycler specific RT RNA QC PCR Arrays Important points before starting E Ensure that the RT SYBR Green Mastermix and the
45. nscription reaction is apparent No action is needed m If this value is greater than 5 there is evidence of impurities that may have inhibited the reverse transcription reaction See the Troubleshooting Guide page 36 A4 Examine the C values of the positive PCR control well PPC of each sample as follows RT Predictor PCR Array Handbook 10 2012 41 Calculate The value should be 20 2 on each RT Predictor PCR Array and should not vary by more than 2 cycles between different samples and RT Predictor PCR Arrays being compared m Larger differences values between samples indicate the presence of PCR amplification inhibitors This means that the RNA samples require further purification An average value of that is consistently greater than 22 for all samples may indicate a problem with the cycling conditions or may simply be indicative of the relative sensitivity of your instrument See the Troubleshooting Guide page 36 A5 Calculate the AC for each pathway specific signature gene in each sample using the C values for the signature gene Signature and the housekeeping genes used for normalization HKG Use the formula AC Ce HKG Note The expression level of the housekeeping genes chosen for normalization must not be influenced by the experimental conditions If one or more such housekeeping genes are not consistent between samples these house
46. onditions or may simply be indicative of the relative sensitivity of your instrument See the Troubleshooting Guide page 36 A5 Calculate the AC for each pathway specific signature gene in each sample using the values for the signature gene Signature and the housekeeping genes used for normalization HKG Use the formula AC C we HKG RT Predictor PCR Array Handbook 10 2012 43 Note The expression level of the housekeeping genes chosen for normalization must not be influenced by the experimental conditions If one or more such housekeeping genes are not consistent between samples these house keeping genes can be excluded from analysis of the average of HKG C values in the equation above If an appropriate housekeeping gene has not been previously identified use the average value of all housekeeping genes A6 When biological and or technical replicates are performed calculate the average AC value of each gene each well across those replicate arrays for each treatment group A7 Calculate the AAC for each gene across 2 samples of RT Predictor PCR Arrays or groups of samples Use the formula AC group 2 AC group 1 where group 1 is the control sample or group of control samples and group 2 is the experimental sample or group of experimental samples A8 Calculate the fold change for each gene from group 1 to group 2 as 2 Detailed mathematical explanation of
47. primer assays for 4 sets of genes and each set contains 16 pathway specific signature genes 5 housekeeping genes one genomic DNA control one reverse transcription controls and one positive PCR controls Figure 1 RT Predictor PCR Arrays in 384 well plates contain 16 replicate primer assays for each set of genes comprising 16 pathway specific signature genes 5 housekeeping genes one genomic DNA control one reverse transcription control and one positive PCR control Figure 2 RT Predictor PCR Arrays in Rotor Disc 100 format contain primer assays for 4 sets of genes Each set contains 16 pathway specific signature genes 5 housekeeping genes one genomic DNA control one reverse transcription control and one positive PCR control Wells 97 100 of the Rotor Disc 100 do not contain assays Figure 3 During the procedure master mix is added to these wells for balance but the wells are not used for analysis Definitions of controls in RT Predictor PCR Arrays Assays for 5 housekeeping genes included in the arrays enable normalization of data The genomic DNA control GDC is an assay that specifically detects nontranscribed genomic DNA contamination with a high level of sensitivity The reverse transcription control RTC is an assay that tests the efficiency of the reverse transcription reaction performed with the RT First Strand Kit by detecting template synthesized from the kit s built in external RNA control The positive PCR control
48. protocolfiles php Table 8 Cycling conditions for Rotor Gene cyclers Cycles Duration Temperature Comments 10 min 95 C HotStart DNA Taq Polymerase is activated by this heating step 40 15 5 95 30 5 60 Perform fluorescence dota collection re Insert the RT Predictor PCR Array into the Rotor Disc 100 Rotor and secure with the Rotor Disc 100 Locking Ring Start the run For detailed instructions see the Rotor Gene Q User Manual Calculate the threshold cycle for each well using the real time cycler software To define the baseline select Dynamic Tube RT Predictor PCR Array Handbook 10 2012 33 10 11 34 default analysis setting to ensure that the average background of each well is determined just before amplification commences Optional Select Ignore First Fluorescent signal from the initial cycles may not be representative of the remainder of the run Thus better results may be achieved if the initial cycles are ignored Up to 5 cycles can be ignored Optional Select Noise Slope Correction Selection of this option can improve data whose baseline initial cycles is noticeably sloped Noise Slope Correction improves the data when raw data backgrounds are observed to slope upward or downward before the takeoff point Note Ensure that the settings are the same across all RT Predictor PCR Array runs in the same analysis Manually define the threshold by usi
49. r PCR Array results it is important to avoid contamination of the assay with foreign DNA Any DNA contamination will artificially inflate the SYBR Green signal yielding skewed gene expression profiles and false positive signals that affect accurate determination of pathway activity regulation The most common sources of DNA contamination are the products of previous experiments spread into the air of the working environment To set up and maintain a working environment free of DNA contamination follow the guidelines below Wear gloves throughout the procedure Use only fresh PCR grade reagents water and labware tips and tubes Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate the PCR workspace and labware pipettor barrels tube racks etc before each use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 1096 bleach to chemically inactivate and degrade any DNA M Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any labware tips or tubes containing PCR products or other DNA treat with 1096 bleach Do not remove the RT Predictor PCR Array from its protective sealed bag until immediately before use Do not leave labware tubes and tip boxes exposed to the air for long periods of time Do not open any previously run and stored RT P
50. r up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Larger kit sizes available please inquire 48 RT Predictor PCR Array Handbook 10 2012 Trademarks QIAGEN QlAamp QIAzol GlAgility Rotor Disc QIAGEN Group Fluidigm BioMark Fluidigm Corp PAXgene PreAnalytiX GmbH Roche LightCycler TaqMan Roche Group Applied Biosystems ROX StepOnePlus ViiA Applera Corporation or its subsidiaries Eppendorf Mastercycler Eppendorf AG Stratagene Mx3005P Mx3000P Mx4000 Stratagene Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ MyiQ Bio Rad Laboratories Inc Excel Microsoft Inc LabChip Caliper Technologies Corp Agilent Agilent Technologies Inc FACS Becton Dickinson and Company SmartCycler Cepheid DNA free Ambion Inc SYBR Life Technologies Corporation TRIzol RNAzol Molecular Research Center Inc Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the RT Predictor PCR
51. redictor PCR Array Removing the thin wall 8 cap strips or the adhesive film from PCR arrays releases PCR product DNA into the air where it may affect the results of future real time PCR experiments RNA preparation quantification and quality control High quality RNA is essential for obtaining good real time PCR results The most important prerequisite for any gene expression analysis experiment is consistently high quality RNA from every experimental sample Residual traces of proteins salts or other contaminants may degrade the RNA or decrease the efficiency of enzyme activities necessary for optimal reverse transcription and real time PCR performance 18 RT Predictor PCR Array Handbook 10 2012 Recommended RNA preparation methods High quality total RNA for your real time PCR experiment should be prepared using one of the methods described below depending on the biological sample For optimal results RNA samples should be suspended in RNase free water Do not use DEPC treated water Cultured cells We recommend the RNeasy Mini Kit cat no 74104 for RNA purification from cultured cells It is important to perform the on column DNase digestion step described in the RNeasy Mini Handbook using the RNase Free DNase Set cat no 79254 Tissue samples We recommend the RNeasy Microarray Tissue Mini Kit cat no 73304 including the optional on column DNase digestion step described in the RNeasy Microarray Tissue Handbook u
52. score of pathway activity EJ Summary of pathway activity scores Sample Pathway activity score P value A 0 926092 0 01 B 0 904272 0 01 C 0 142092 0 93 Figure 7 RT Predictor PCR Array data analysis output Calculation of pathway activity regulation in experimental samples based on expression levels of pathway specific signature biomarker genes with a classification algorithm The negative score of sample A indicates the repression of pathway activity in sample A as compared to the control In contrast the positive score of sample B indicates pathway activity is activated in sample B as compared to the control sample The pathway activity of sample C remains unchanged 38 RT Predictor PCR Array Handbook 10 2012 Summary table of pathway activity prediction scores Median pathway activity prediction score of each sample is shown in a table below the box and whisker plot A statistical P value will be provided for each sample The P value is calculated based on comparison of the experimental sample to untreated samples of the signaling pathway or negative control treatment samples of the toxic pathway in the training data set Samples with P 0 05 as well as a pathway activity score 20 3 or lt 0 3 indicate significant change of pathway activity as compared to control The AAC values of signature biomarker genes between experimental and control samples are used to derive the pathway activity probability score
53. sing the RNase Free DNase Set cat no 79254 Whole blood samples We recommend the PAXgene Blood RNA Kit cat no 762174 for preparation of total RNA from whole blood samples Alternatively the QlAamp RNA Blood Mini Kit cat no 52304 can also be used for this purpose Total RNA isolated using a phenol based method Total RNA from any biological source material prepared using a phenol based method e g QIAzol Lysis Reagent TRIzol Reagent RNAzol Reagent should be further purified using the RNeasy Mini Kit It is important to perform the on column DNase digestion step described in the RNeasy Mini Handbook Other biological samples Refer to the existing literature to find protocols for high quality RNA purification from other biological samples or contact QIAGEN Technical Service RNA quantification and quality control For best results from the RT Predictor PCR Array all RNA samples should also demonstrate consistent quality according to the criteria described below In addition as some contaminants are difficult to detect by simply looking at RNA integrity and can be missed by UV spectrophotometry it is essential to choose RT Predictor PCR Array Handbook 10 2012 19 an appropriate RNA purification method for your biological sample as described on page 19 23 Concentration and purity determined by UV spectrophotometry The concentration and purity of RNA should be determined by measuring the absorbance in
54. tegrity Agilent Bioanalyzer electropherogram of high quality total RNA showing sharp peaks for the 185 left and 285 right ribosomal RNA Due to high quality of the RNA peaks do not have shoulders especially to the left of each peak Agarose gel electrophoresis shows sharp bands especially at the bottom of each band for 28S and 185 ribosomal RNA RT RNA QC PCR Array optional The RT RNA QC PCR Array is particularly useful for researchers who are unsure of their RNA purification technique The RT RNA QC PCR Array and the RT First Strand Kit sold separately test for a number of RNA quality control parameters including High and low housekeeping gene expression levels E Reverse transcription and polymerase chain reaction efficiency M Genomic and general DNA contamination For further details consult the RT RNA QC PCR Array Handbook Genomic DNA contamination Eliminating genomic DNA contamination is essential for obtaining optimal analysis of pathway regulation using the RT Predictor PCR Array The genomic DNA control in each RT Predictor PCR Array specifically tests for genomic DNA contamination in each sample during each run A genomic DNA control threshold cycle value of less than 35 indicates the presence of a detectable amount of genomic DNA contamination that should be addressed To remove any residual contamination from your RNA samples we strongly recommend RNA purification using the RNeasy Mini Kit includi
55. to first strand cDNA using the RT First Strand Kit Next the cDNA is mixed with an appropriate RT SYBR Green Mastermix This mixture is aliquoted into the wells corresponding to each sample on the RT Predictor PCR Array PCR is performed and finally pathway activity regulation is determined using data from the real time cycler and the classification methods derived from our training data with experimentally verified pathway manipulation ctm H M H PH M M H RT Predictor PCR Array Handbook 10 2012 15 2 T Isolate RNA from research samples cells tissues and or blood lt m d b Convert total RNA to cDNA with RT First Strand Kit Control Experimental Add cDNA to RT SYBR Green qPCR Mastermix aliquot across RT Predictor PCR Array Generate dato in any PCR instrument Interpret your data and publish your results Figure 4 RT Predictor PCR Array procedure RNA quality control using an RT RNA QC PCR Array The RT RNA QC PCR Array is designed to assess the quality of 12 RNA samples simultaneously before gene expression analysis using RT Predictor PCR Arrays Use of the RT RNA QC PCR Array provides complete confidence in gene expression analysis results by enabling exclusion of substandard samples prior to analysis with RT Predictor PCR Arrays For further details consult the RT RNA QC PCR Array Handbook Description of protocols This
56. uality control 18 Recommended RNA preparation methods 19 RNA quantification and quality control 19 Genomic contamination 21 Starting RNA amounts 22 Protocols cDNA Synthesis Using the RT First Strand Kit 23 Real Time PCR for RT Predictor PCR Arrays Formats A D E F G 25 Real Time PCR for RT Predictor PCR Arrays Format R 32 Troubleshooting Guide 36 Appendix A Data Analysis 38 Derivation of pathway activity probability score based on signature biomarker gene expression between experimental and control samples 38 Classification model and signature biomarker genes derivation from training data set 40 RT Predictor PCR Array Handbook 10 2012 3 Principle of AAC calculation between control and experimental groups for 96 well and 384 well formats 41 Principle of AAC calculation between control and experimental groups for Rotor Disc formats 43 Detailed mathematical explanation of AAC data analysis method 44 References 46 Ordering Information 47 4 RT Predictor PCR Array Handbook 10 2012 Kit Contents RT Predictor PCR Array Format A 96 well plate containing dried assays 24 Optical Thin Wall 8 Cap Strips 12 per 96 well plate 288 Handbook 1 Suitable for use with the following real time cyclers Applied Biosystems models 5700 7000 7300 7500 7700 7900HT ViiA 7 96 well block Bio Rad models iCycler iQ 5 MyiQ MyiQ2 Bio Rad MJ Research Chromo4 Eppendorf Mastercycler ep
57. ult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www qgiagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s Quality Management System each lot of RT Predictor PCR Arrays RT First Strand Kits and RT SYBR Green Mastermixes is tested against predetermined specifications to ensure consistent product quality RT Predictor PCR Array Handbook 10 2012 11 Introduction Real time RT PCR is a highly sensitive and reliable method for gene expression analysis Its wide dynamic range makes real time RT PCR the preferred choice for the simultaneous quantification of multiple genes in the same sample RT Predictor PCR Arrays are designed to analyze a panel of genes related to a biological pathway to determine intracellular status of biological function such as signaling and toxic pathway activity The array along with classification algorithm indicates whether the pathway is activated repressed or whether activity remains unchanged in an experimental sample as compared to a control sample For each pathway regulation of pathway activity is determined from the expression levels of its signature biomark
58. y Handbook 10 2012 31 Protocol Real Time PCR for RT Predictor PCR Arrays Format R Important points before starting Ensure that RT Predictor PCR Array Format R and RT SYBR Green FAST Mastermix are used with a Rotor Gene cycler The format of the RT Predictor PCR Array is indicated by the last letter of the catalog number For accuracy and precision ensure that micropipettors are calibrated before beginning the protocol Be sure not to introduce bubbles into the wells of the RT Predictor PCR Array when pipetting Do not use DEPC treated water Use high quality nuclease free water If precipitates are present in the Mastermix tubes warm the reagents at 42 for 1 min and vortex briefly to dissolve Repeat if necessary Procedure 1 Briefly centrifuge the RT SYBR Green ROX FAST Mastermix water and cDNA synthesis reaction 10 15 s to bring the contents to the bottom of the tubes Note As the RT SYBR Green ROX FAST Mastermix contains HotStart DNA Taq Polymerase that is active only after heat activation reactions can be prepared at room temperature 15 25 C 2 Prepare the PCR components mix in a 1 5 ml tube as described in Table 7 Table 7 PCR components mix Array format Rotor Disc 100 2x RT SYBR Green ROX FAST Mastermix 300 ul cDNA synthesis reaction 40 ul RNase free water 260 ul Total volume 600 Note This provides an excess volume of 120 ul to allow for pipetting

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