User Manual RT FFPE PreAMP cDNA Synthesis Kit
Contents
1. is required to activate the HotStart Taq DNA polymerase 95 C 10 min 40 cycles of 95 C 15 sec and 60 C 60 sec Program the real time thermal cycler to detect and record the SYBR Green signal from every reaction during the annealing step of each cycle Technical Support 888 503 3187 US 301 682 9200 11 RT FFPE PreAMP cDNA Synthesis Kit VI Detailed Protocol Please read through this entire protocol before beginning your experiment RNA samples are very sensitive to RNase digestion therefore wear gloves and maintain an RNase free work area while performing this protocol Important Note Please note that each RT FFPE PreAMP Primer Mix is specific to a catalogued RT Profiler PCR Array and can only be used for the intended PCR Array Please also verify that the lot number of your Primer Mix is compatible with that of the RT Profiler PCR Arrays you are going to use Otherwise the PreAMP process may not work optimally If the Primer Mix and the PCR Arrays have been purchased at different times please check with our Customer service 1 888 503 3187 to ensure their compatibility NOTE Master Mix and First Strand Synthesis Considerations The performance of our RT FFPE PreAMP cDNA Synthesis Kit is only guaranteed with our RT FFPE PreAMP Primer Mix and our RT2Profiler PCR Arrays and RT qPCR SYBR Green Master Mixes In addition the RT PreAMP PCR Master Mix in this kit is specially formulated for multiplex PCR2. FFPE PreAMP cDNA Synthesis Kit Il Materials Provided This kit includes enough of the following reagents for 12 RNA samples A RT First Strand cDNA Synthesis Components enough for 12 20 ul RT reactions One 1 tube of GE 5X gDNA Elimination Buffer One 1 tube of BC3 5X Reverse Transcription Buffer 3 One 1 tube of RE CDNA Synthesis Enzyme Mix One 1 tube of RI RNase Inhibitor One 1 tube of P2 Primer and External Control Mix One 1 tube of RNase free H20 000000 B RT PreAMP components o One 1 tube of RT PreAMP PCR Master Mix PA 030 containing 600ul of 2X solution and enough for four 25 ul standard reactions per sample for 12 samples 48 reactions o One 1 tube containing 96ul Side Reaction Reducer SR1 enough for four standard reactions per sample for 12 samples 48 reactions Storage Conditions All components included in this kit are shipped on dry ice or blue ice packs and must be stored at 20 C upon receipt When stored properly at 20 C their quality is guaranteed for 6 months Note Ensure that you do not contaminate the 2X RT PreAMP PCR Master Mix and avoid repeated freezing and thawing by dividing into aliquots containing the amount of cocktail necessary for the number of reactions you are preparing each day The rest of the cocktail should be kept in storage away from any sources of template DNA Version 1 0 lll Additional Materials Required A RNA Isolation Kit See Page
3. Our RT First Strand cDNA synthesis system in this kit comes with a built in external RNA control template that would be detected by the Reverse Transcription Control RTC tests in the RT Profiler PCR Arrays This allows the detection of any presence of inhibitors of reverse transcription ensuring the efficiency of the first strand cDNA synthesis reactions o Pre Amplification of cDNA for pathway specific genes Each first strand cDNA synthesis reaction from 100ng to 1ug of total RNA can be amplified by 4 different sets of PCR Array specific primer mixes allowing gene expression analysis on as many as four different pathways You can also create your own primer mixes using our RT qPCR Primer Assays with only genes that you are interested in for up to ten targets The Side Reaction Reducer eliminates the residual primers after pre amplification making the pre amplified cDNA ready for PCR Array or individual qPCR assay analysis To complete the PCR Array procedure mix the amplified templates with one of our instrument specific and ready to use RT qPCR SYBR Green Master Mixes Aliquot the mixture into each well of the same plate containing pre dispensed gene specific primer sets Perform PCR and finally determine relative expression with your real time instrument and the AAC method Important Note Each RT FFPE PreAMP Primer Mix is PCR Array specific and can only be used for the specified RT Profiler PCR Arrays The first strand synthesis comp
4. and polymerase chain reaction efficiency e Genomic and general DNA contamination Technical Support 888 503 3187 US 301 682 9200 15 RT FFPE PreAMP cDNA Synthesis Kit The RNA QC PCR Arrays are particularly useful for researchers who are unsure of their RNA isolation technique Follow the recommendations for the use and interpretation of the RT RNA QC PCR Array found in its User Manual 4 Amount Considerations The RT FFPE PreAMP cDNA Synthesis Kit generates sufficient templates for gene expression analysis in RT Profiler PCR Arrays with as little as 100 ng or as much as 1 ug total RNA input into each first strand cDNA synthesis reaction prior to pre amplification step Each RT reaction allows the user to perform as many as four RT PreAMP PCR reactions and four RT Profiler PCR Arrays However 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 that is genes expressed in the linear dynamic range of the method Lower amounts of input total RNA yield a smaller number of positive calls and increase false negative calls The RT First Strand cDNA Synthesis components in this kit are optimized for the subsequent RT PreAMP procedures and maximizes the number of positive calls at low amounts 100 ng of total RNA For s
5. by heat inactivation at 95 C for 5 min c Add immediately 28 ul of RNase DNase free H20 to each 27 ul of PreAMP PCR reaction Mix well d Hold the finished PreAMP PCR reaction on ice until the next step Step 7 or store overnight at 20 C 7 Perform Real time RT qPCR Primer Assays NOTE The use of SABiosciences RT qPCR Master Mixes is critical for obtaining the most accurate results from the PCR Array Be sure to use the correct master mix for your instrument before continuing with this protocol See Page 8 NOTE The accuracy and precision of your pipetting determine the consistency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure to not introduce any bubbles into the wells of the PCR Array Experimental Samples a To insure the consistency of your results and that each experimental sample yields a reliably detectable Ct value we recommend setting up duplicate or triplicate reactions for each template b For every experimental sample prepare one set of reactions for every gene of interest and for a single housekeeping gene or a set of housekeeping genes to normalize your raw data Choose housekeeping gene s known to not change their expression under your experimental conditions c Positive and Negative Controls o Prepare a positive control reaction using template known to represent the genes of interest such as template generated from SABioscience
6. on ice immediately for at least one minute 2 Prepare the RT Cocktail RT Cocktail 1 reaction BC3 5X Reverse Transcription Buffer 3 4 ul P2 Primer amp External Control Mix 1 ul RE cDNA Enzyme Synthesis Mix 1 ul RI RNase Inhibitor 1 ul RNase free H20 3 i Scale up the volume for each of the RT Cocktail reagents accordingly when multiple reactions are carried out to minimize pipetting variations 3 First Strand cDNA Synthesis Reaction a Add 10 ul of RT Cocktail from Step B 2 to each 10 ul Genomic DNA Elimination Mixture from Step B 1 b Mix well but gently with a pipettor Spin the tubes briefly to remove any air bubbles and collect all the liquid to the bottom c Incubate at 37 C for exactly 30 min and then immediately stop the reaction by heating at 95 C for 5 minutes d Hold the finished First Strand cDNA Synthesis Reaction on ice until the next step Step C or store overnight at 20 C Technical Support 888 503 3187 US 301 682 9200 17 RT FFPE PreAMP cDNA Synthesis Kit C Pre Amplification of cDNA Target Templates NOTE The use of SABiosciences RT FFPE PreAMP Primer Mix Cat No PFH XXXX PFM XXXX PFR XXXxX is critical for a successful multiplex PCR pre amplification of your cDNA and for obtaining the best results from the PCR Array Be sure to check the label to verify that you have the correct pathway and lot number of the RT FFPE PreAMP Primer Mix for the
7. possible double check the A260 A280 and A260 A230 ratios of your RNA samples in RNase free Tris pH 8 0 buffer If necessary re purify your RNA samples with a spin column based clean up method such as SABiosciences RT gPCR Grade RNA Isolation Kit PA 001 3 Improving Poor PCR Amplification Efficiency Different instruments have different levels of sensitivity If an average C value of 20 2 is difficult to obtain for your instrument the observed average C value should be acceptable as long as it does not vary by more than two cycles between PCR Arrays being compared Be sure that the initial heat activation step at 95 C had been lengthened to 10 minutes from the shorter time in the default program Be sure that all other cycle parameters also have been correctly entered according to the recommendations in the User Manuals of this kit and the RT Profiler PCR Array Also double check the quality of your RNA as described in Evidence of Poor Reverse Transcription Efficiency above 26 Version 1 0 B Frequently Asked Questions 1 Shall use C values that are equal to or greater than 35 Any C values gt 35 is considered a negative call Due to the inclusion of the pre amplification step for the assays showing C values greater than 35 the expression level of these genes will be too low to be reliably quantified Consider removing these data points from the rest of the results 2 What is the range of RNA am
8. pre amplification step for the assays showing C values greater than 35 the expression level of these genes will be too low to be reliably quantified Consider removing these data points from the rest of the results 2 Examine the threshold cycle values of the control wells a Genomic DNA Control GDC i Calculate C 9 ii If the value is greater than 31 then the level of genomic DNA contamination is too low to affect gene expression profiling results No action is needed If the value is less than 31 then genomic DNA contamination is evident See the Troubleshooting and FAQ section b Reverse Transcription Control RTC Any impurities in your RNA sample that affect the reverse transcription of the RT First Strand cDNA Synthesis built in external RNA control in this kit also affect the reverse transcription of your messages of interest i Calculate AC AVG C AVG CPFY ii If this value is less than 7 then no inhibition is apparent iii If this value is greater than 7 then evidence of impurities that inhibited the reverse transcription phase of the procedure is evident See the Troubleshooting and FAQ section 20 Version 1 0 c Positive PCR Control PPC Any impurities in your RNA sample that affect the PCR amplification of the positive control also affect the PCR amplification for your messages of interest i The average C P value should be 20 2 on each PCR Array and should not vary by mo
9. sample is divided by the normalized expression of the same GOI in the control sample g AClexpt a Acieontro 2 Where AAC is equal to AC expt AC control The complete calculation is as follows ZACH expt giC0N CHKCH expt Z expt ort Ze control ge A e iol 2 contra 7 ACAAKG control 22 Version 1 0 Vil Alternative PreAMP Protocol for Individual RT qPCR Primer Assays 1 Perform First Strand cDNA Synthesis as in Part VI Step B 2 Prepare the PreAMP Primer Mix To prepare 1 mL of the PreAMP primer mix for your selected gene s up to ten genes using SABiosciences RT qPCR Primer Assays pipet 40 ul of each primer set into a 1 5 mL microcentrifuge tube and add DNase RNase free H20 according to the table below Vortex to mix well and spin down the tube briefly 2 an i j uM rAssay DNase RNase free H20 1 gene 1 gene x 40 ul each 40 ul 960 ul 2 genes 2 genes x 40 ul each 80 ul 920 ul 3 genes 3 genes x 40 ul each 120 ul 880 ul 4genes 4genes x 40 ul each 160 ul 840 ul 9 genes 5 genes x 40 ul each 200 ul 800 ul 6 genes 6 genes x 40 ul each 240 ul 760 wl 7 genes 7 genes x 40 ul each 280 ul 720 ul 8 genes 8 genes x 40 ul each 320 ul 680 ul 9 genes 9 genes x 40 ul each 360 ul 640 ul 10 genes 10 genes x 40 ul each 400 ul 600 ul Note One mL of the primer mix is sufficient for 130 PreAMP reactions 3 Thaw the RT PreA
10. 13 for specific recommendations B For Gene Expression Analysis using RT Profiler PCR Arrays a RT FFPE PreAMP Primer Mix PFH XXXX PFM XXXX PFR XXXX MANDATORY for a Successful and Unbiased Pre Amplification of your samples each primer mix is specific to a cataloged RT Profiler PCR Array Check the label to verify that you have the correct pathway specific RT PreAMP Primer Mix for the PCR Arrays you are performing Please also verify that the lot number of your Primer Mix is compatible with that of the RT Profiler PCR Arrays you are going to use We strongly recommend the Primer Mix and the PCR Arrays to be purchased together whenever possible If the Primer Mix and the PCR Arrays have been purchased at different times please check with our Customer service 1 888 503 3187 to ensure their compatibility b SABiosciences RT Profiler PCR Array The PCR Arrays are available in six different plate formats each tailored to a specific subset of real time PCR instruments and associated blocks Formats A C D and F are 96 well plates while Formats E and G are 384 well plates Technical Support 888 503 3187 US 301 682 9200 7 RT FFPE PreAMP cDNA Synthesis Kit Format For Real Time Instruments Plate All ABI standard blocks 7000 7300 7500 7700 7900 Bio Rad iCycler MyiQ iQ5 A Bio Rad MJ Research Chromo 4 FPW Eppendorf RealPlex Stratagene Mx3005p Mx3000p ABI 7500 and 7900HT FAST 96 well blocks ABI Step
11. Arrays can only be used in 96 well and 384 well real time PCR instruments PCR Arrays can not be used in the Cepheid SmartCycler the Roche LightCycler 2 0 or the Corbett Research Rotorgene 0 2 mL individual or 8 tube strip PCR tubes with caps Calibrated P2 P20 P200 and P1000 Single Channel Pipettors Calibrated Multi Channel Pipettor RNase DNase free pipette tips and tubes D NPE w IV Complementary Products XpressRef Universal Total RNA Universal RNA to control PCR conditions is available from the following species Human XpressRef Universal Total RNA Cat No GA 004 Mouse XpressRef Universal Total RNA Cat No GA 005 Rat XpressRef Universal Total RNA Cat No GA 006 Version 1 0 V Quick Protocol 1 10 Before starting the experiment make sure both the pathway and the lot number of your RT FFPE PreAMP Primer Mix are compatible with those of your RT Profiler PCR Array Refer to page 4 and page 11 for details Perform first strand cDNA synthesis as follows Add 2 ul of GE to 8 ul of RNA 100ng 1yWg Incubate at 37 C for 15 min and immediately chill on ice Mix a master mix for the RT reaction as below For 1 reaction BC3 4 ul RE 1 ul RI 1 ul P2 1 yl RNase free H2O 3 ul Add 10 ul of the RT master mix to 10 ul GE treated RNA Incubate at 37 C for 30 min and heat at 95 C for 5 min Chill on ice or store at 20 C until use For the normal standard reaction mix the following co
12. MP PCR Master Mix PA 030 at room temperature If precipitates are observed warm the reagents at 42 C for 1 min and vortex briefly to dissolve Repeat the process if necessary 4 Prepare the PreAMP PCR Cocktail RT PreAMP PCR Cocktail 1 reaction RT PreAMP PCR Master Mix PA 030 2X Solution 12 5 ul PreAMP Primer Mix 7 5 ul Scale up the volume for each of the PreAMP PCR Cocktail reagents accordingly when multiple reactions are carried out to minimize pipetting variations 5 PreAMP PCR Reaction a Pipet 5 ul of the First Strand cDNA Synthesis Reaction into a 0 2 ml PCR tube Then add 20 wl of the PreAMP PCR Cocktail from Step 4 b Mix well but gently with a pipettor Spin the tubes briefly to remove any air bubbles and collect all the liquid to the bottom c Perform 8 cycles of PCR in a thermal cycler NOTE The 10 min step at 95 is required to activate the HotStart Taq DNA polymerase 95 C 10 min 8 cycles of 95 C 15 sec and 60 C 2 min 4 C forever Technical Support 888 503 3187 US 301 682 9200 23 RT FFPE PreAMP cDNA Synthesis Kit 6 Treatment with the Side Reaction Reducer a Take out the tubes from the thermal cycler and put on ice Add 2 ul of the Side Reaction Reducer SR1 to each pre amplified reaction Mix well but gently with a pipettor Spin the tubes briefly to remove any air bubbles and collect all the liquid to the bottom b Incubate at 37 C for 15 min followed
13. OnePlus JPEN Bio Rad CFX96 D Opticon and Opticon 2 MJ Research NE Stratagene Mx4000 E ABI 7900HT 384 well block 384 well F Roche LightCycler 480 96 well block 96 well G Roche LightCycler 480 384 well block 384 well NOTE The format of the PCR Array is indicated by the last letter of the catalog number Be sure that you have the correct PCR Array format for your instrument before starting the experiment The 96 well PCR Arrays Formats A C D and F are shipped in sets of two 2 or twelve 12 while the 384 well PCR Arrays Formats E and G are shipped in sets of four 4 Each PCR Array shipment includes the arrays and either twelve 12 optical thin wall 8 cap strips Formats A and D or one 1 optical adhesive film Formats C E F and G per array Version 1 0 OR RT qPCR Primer Assay s PPH XXXXX PPM XXXXX PPR XXXXX To make your own primer mix for up to 10 individual genes of your choice order the RT qPCR Primer Assays specific to your selected genes and species form our catalog Follow the instructions on Alternative PreAMP Protocol for Individual Primer Assays on page 22 to make the primer mix and perform qPCR Assays SABiosciences RT qPCR Master Mix MANDATORY for a Complete and Successful Experiment Be sure to pick the correct one for the instrumentation in your laboratory 1 96 Well PCR Arrays RT SYBR Green ROX qPCR Master Mix Specifically designed for All ABI and Stratagene Instrumenta
14. PCR Arrays you are performing See pg 4 and pg 11 for details NOTE The accuracy and precision of your pipetting determine the consistency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure to not introduce any bubbles into the wells of the PCR tubes 1 Thaw the RT PreAMP PCR Master Mix PA 030 and RT FFPE PreAMP Primer Mix at room temperature If precipitates are observed warm the reagents at 42 C for 1 min and vortex briefly to dissolve Repeat the process if necessary 2 Prepare the PreAMP PCR Cocktail RT PreAMP PCR Cocktail 1 reaction RT PreAMP PCR Master Mix PA 030 2X Solution 12 5 ul RT FFPE PreAMP Primer Mix each specific for a 75 ul RT Profiler PCR Array es Scale up the volume for each of the PreAMP PCR Cocktail reagents accordingly when multiple reactions are carried out to minimize pipetting variations 3 PreAMP PCR Reaction a Pipet 5 ul of the First Strand cDNA Synthesis Reaction from Step B 3 into a 0 2 ml PCR tube Then add 20 ul of the PreAMP PCR Cocktail from Step C 2 b Mix well but gently with a pipettor Spin the tubes briefly to remove any air bubbles and collect all the liquid to the bottom c Perform 8 cycles of PCR in a thermal cycler NOTE The 10 min step at 95 is required to activate the HotStart Taq DNA polymerase 95 C 10 min 8 cycles of 95 C 15 sec and 60 C 2 min 4 C for
15. PreAMP cDNA Synthesis Kit includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any primer pair mix to modify kit components for resale or to use RT Profiler PCR Array to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppels is granted U S patents may cover certain isolated DNA sequences included in the RT Profiler PCR Array Presently it is not clear under U S laws whether commercial users must obtain licenses from the owners of the rights to these U S patents before using RT Profiler PCR Array Patents are pending on the RT Profiler PCR Array System technology itself Technical Support 888 503 3187 US 301 682 9200 3 I Background and Introduction Annotated tissue archives with known clinical outcomes are abundant in the form of formalin fixed and paraffin embedded states Interests in using these samples for retrospective molecular studies have grown over the past decade with the development of extraction methods to unlock nucleic acids from them However analysis of RNA from those samples present many challenges The quality of RNA is often compromised during sample handling prior to fixation formalin fixation storage over time and isolation procedures RNA in FFPE samples is likely to be fragmented and chemically modified by forma
16. based pre amplification with our RT FFPE PreAMP Primer Mix for optimal results without introducing bias The chemically modified and tightly controlled HotStart enzyme and other proprietary chemical components in our RT qPCR Master Mixes uniquely provide more accurate SYBR Green results by preventing the amplification of primer dimers and other non specific products They also help ensure high amplification efficiencies even for those genes that are the most difficult to amplify When we test other sources of enzymes with our PCR Arrays we frequently see primer dimers and other non specific products that confound SYBR Green based real time PCR detection Because each instrument uses a different reference dye to normalize their optics be sure that you use the correct master mix for the instrumentation in your laboratory Our RT First Strand cDNA Synthesis components in this kit include a proprietary buffer to eliminate any residual genomic DNA contamination in your RNA samples before it can be amplified into secondary products that would otherwise cause false positive signals The Reverse Transcription Controls RTC on our RT Profiler PCR Array can only be evaluated with the built in external RNA control of our RT First Strand cDNA Synthesis components These controls do not yield results when used with other sources of reverse transcriptases or first strand synthesis kits The buffer components and the magnesium concentration in our RT First S
17. e archiving procedure and may depend on the age of the block and other less tangible variables 1 Standard fixation recommendations for the eventual purposes of gene expression analysis call for fixing tissue samples of thickness no more than 5 mm for 14 to 24 hours in 4 to 10 neutralized formalin as quickly as possible after surgical removal Longer fixation times and longer periods of time from surgery to fixation lead to more severe RNA fragmentation and chemical modification resulting in poor performance in downstream assays It is also highly recommended to completely dehydrate the samples prior to embedding in paraffin Previously archived samples may not have been processed in this fashion and the details of how the process was actually performed may not be available However with appropriate FFPE RNA isolation procedures which can effectively reverse chemical cross linking gene expression analysis may still be performed Recommended RNA Preparation Methods For best results use SABiosciences RT FFPE RNA Extraction Kit Catalog PF 023 The RT FFPE RNA Extraction Kit uses an optimized RNA isolation protocol that reverses more cross links than other methods and removes salts metabolites and macromolecular cellular component and other contaminants that would otherwise inhibit the qRT PCR processes making RNA more available and ready for cDNA template synthesis and qRT PCR We have tested a few other commercial FFPE RNA isolatio
18. e e e a re oe SABioscienceS Baran 22769 Ha amou wra biomol info bio www bio Phon ae vi 8532600 r 0800 2466651 D Fax 49 40 2A r 0800 2466652 D User Manual C RT FFPE PreAMP cDNA Synthesis Kit Synthesis and Pre amplification of First Strand cDNA from RNA of Formalin Fixed Paraffin Embedded Samples for Gene Expression Profiling with RT Profiler PCR Arrays See Purchaser Notification for limited use license and warranty information page 3 Part 1046A Version 1 0 3 24 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA RT FFPE PreAMP cDNA Synthesis Kit Synthesis and Pre amplification of First Strand cDNA from RNA of Formalin Fixed Paraffin Embedded Samples for Gene Expression Profiling with RT Profiler PCR Arrays User Manual F C I N b C 07 BIOMOL GmbH s Waidmannstr 35 Opera og NAMOEN GON amas CY biomol www biomol de Ordering and Technical Service Contact Information Phor 2940 8532600 or 0800 2466651 D e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www SABiosciences com e E MAIL order SABiosciences com to place an order support SABiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or c
19. ever 4 Treatment with the Side Reaction Reducer a Take out the tubes from the thermal cycler and put on ice Add 2 ul of the Side Reaction Reducer SR1 to each pre amplified reaction Mix well but gently with a pipettor Spin the tubes briefly to remove any air bubbles and collect all the liquid to the bottom b Incubate at 37 C for 15 min followed by heat inactivation at 95 C for 5 min c Add immediately 84 ul of RNase DNase free H2O to each 27 ul of PreAMP PCR reaction Mix well d Hold the finished PreAMP PCR reaction on ice until the next step Step D or store overnight at 20 C Version 1 0 D Performing Real Time PCR with RT Profiler PCR Arrays NOTE The use of SABiosciences RT qPCR Master Mixes is critical for obtaining the most accurate results from the PCR Array Be sure to use the correct master mix for your instrument before continuing with this protocol See Page 8 NOTE An incorrectly chosen PCR Array plate format will not properly fit into your real time PCR instrument and its use will damage the instrument Be sure you have the correct PCR Array format for your instrument before continuing with this protocol See Page 7 NOTE The accuracy and precision of your pipetting determine the consistency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure to not introduce any bubbles into the wells of the PCR Array 1 Experimental Coc
20. groups to effectively normalize your results 4 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 5 Calculate the AAC for each gene across two PCR Arrays or groups AAC AC group 2 AC group 1 Where group 1 is the control and group 2 is the experimental 6 Calculate the fold change for each gene from group 1 to group 2 as 2 AAC OPTIONAL f the fold change is greater than 1 then the result may be reported as a fold up regulation If the fold change is less than 1 then the negative inverse of the result may be reported as a fold down regulation The fold change ratios may also be reported as is Technical Support 888 503 3187 US 301 682 9200 21 RT FFPE PreAMP cDNA Synthesis Kit NOTE Detailed Mathematical Explanation of AAC 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 fee To normalize the expression level of a gene of interest GOI to a housekeeping gene HKG the expression levels of the two genes are divided 9 G60 eee 9 GGON C HKG I _ gat qe To determine fold change in gene expression the normalized expression of the GOI in the experimental
21. h 4 Do not remove the PCR Array plate from its protective sealed bag until immediately ready to use Do not leave lab ware tubes and tip boxes exposed to the air for long periods of time 5 Do not open any previously run and stored PCR Array plate Removing the thin wall 8 cap strips or the adhesive film from PCR Arrays releases PCR product DNA into the air where it will contaminate and confound the results of future real time PCR experiments Technical Support 888 503 3187 US 301 682 9200 13 RT FFPE PreAMP cDNA Synthesis Kit A FFPE Sample Preparation RNA Preparation Quality and Amount Considerations Good quality RNA is ESSENTIAL for obtaining good real time PCR results FFPE samples are amongst the most difficult RNA samples to work with due to the existence of degradation and chemical modifications in those types of samples To obtain the best possible RNA quality from FFPE samples the sample handling and RNA isolation procedures are critical With any isolation methods it is important to remember that the presence of residual traces of proteins salts or other contaminants will either degrade the RNA or decrease the efficiency of if not block completely the enzyme activities necessary for optimal reverse transcription PreAMP PCR and real time PCR performance 1 FFPE Sample Preparation for Gene Expression Analysis The quality of the RNA isolated from FFPE samples will depend on the technique used to execute th
22. ktail Preparation Plate Format 96 well 384 well A C D amp F E amp G 2X SABiosciences RT qPCR SYBR Green Master Mix 1275 ul 550 ul Diluted RT PreAMP PCR Reaction 102 ul 102 ul RNase DNase free H20 1173 ul 448 ul NOTE This recipe provides an excess volume of ONLY 140 ul Very carefully add the cocktail to the PCR Array precisely to insure that each well receives the required volume 2 For the rest of PCR Array protocol please follow the instructions from Step C 2 to Step C 6 Performing Real Time PCR with RT Profiler PCR Arrays of the RT Profiler PCR Array User Manual Technical Support 888 503 3187 US 301 682 9200 19 RT FFPE PreAMP cDNA Synthesis Kit E Data Analysis AAC Method Upon completion of PCR Array Run Please access the free PCR Array Data Analysis Web Portal at http www SABiosciences com pcrarraydataanalysis php Note Please check the box in the Readout page for RT PreAMP cDNA Synthesis Kit The PCR Array Data Analysis Web Portal automatically performs the following calculations and interpretation of the control wells upon including threshold cycle data from a real time instrument The PCR Array Data Analysis Web Portal presents the results in a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included 1 Any C value equal to or greater than 35 is considered a negative call Due to the inclusion of the
23. ldehyde with extensive cross linking among protein DNA and RNA Some of those damages on RNA are irreversible and will greatly reduce the amount of templates available for interrogation thus affecting the performance and sensitivity of gene expression measurements In particular FFRE RNA samples are not suitable for downstream applications that require full length and intact RNA Quantitative RT PCR is the preferred method to analyze partially degraded FFPE RNA as it only interrogates a short sequence of RNA for each gene ensuring the higher frequency of RNA fragments that contain the complete target sequence RT FFPE PreAMP cDNA Synthesis Kit and Primer Mixes enhance the sensitivity of RT PCR in expression analysis of pathway or disease focused genes in partially degraded RNA from FFPE samples Our RT PreAMP technology utilizes multiplex PCR based pre amplification to capture and amplify gene specific cDNA target templates from FFPE samples with minimal bias This kit is intended for synthesis followed by pre amplification of first strand cDNA from FFPE RNA samples for gene expression analysis with our RT Profiler PCR Arrays or individual RT qPCR Primer Assays You can prepare enough cDNA from each RNA sample for gene expression analysis on as many as four different pathways Two simple steps are involved in this kit o CDNA first strand synthesis This kit provides enough reagents for synthesizing first strand cDNA from 12 different samples
24. mponents in a PCR tube 12 5 ul 2X RT PreAMP PCR Master Mix PA 030 7 5 ul RT FFPE PreAMP Primer Mix specific for the RT Profiler PCR Array of your choice 5 0 ul undiluted cDNA template of the 20 ul first strand synthesis reaction from Step 2 25 0 ul final volume Perform 8 cycles of PCR in a thermal cycler NOTE The 10 min step at 95 is required to activate the HotStart Taq DNA polymerase 95 C 10 min 8 cycles of 95 C 15 sec and 60 C 2 min 4 C forever Add 2 ul of the Side Reaction Reducer SR1 to each pre amplified reaction and incubate at 37 C for 15 min followed by heat inactivation at 95 C for 5 min Dilute the 27 ul pre amplified templates to 111 ul by adding 84 wl of RNase DNase free H20 Use immediately and keep on ice prior to loading onto the RT Profiler PCR Array or store at 20 C until use For use in the RT Profiler PCR Array mix well the following components in 15 mL conical tube 1275 ul 2X SABiosciences RT qPCR SYBR Green Master Mix Note Use the appropriate master mix specific for your real time PCR instrument 102 ul diluted PreAMP PCR reaction from Step 6 1173 u RNase DNase free H2O 2550 ul final volume Add 25 ul of the above Experimental Cocktail to each well of the PCR Array preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips Run the following real time thermal cycler program NOTE The 10 min step at 95
25. n kits including those from Ambion and Qiagen which may also provide similar RNA quality In addition minimizing or eliminating genomic DNA contamination is essential for obtaining optimal gene expression profiling results from real time PCR No currently available purification method can guarantee that RNA is completely free of DNA even when it is not visible on an agarose gel Trace amounts of DNA may still remain after the RT FFPE RNA Extraction procedure You may treat your RNA sample after isolation with a good source of RNase free DNase followed by re purification of the RNA We currently recommend TURBO DNA free kit from Ambion Catalog 1907 In addition the gDNA Elimination Buffer GE in this kit will help remove any and all residual contamination from your RNA samples An 14 Version 1 0 appropriately performed no reverse transcription NRT control during real time PCR is then the best and most accurate test for genomic DNA contamination and should yield real time C values greater than 35 without PreAMP process If these C values are less than 35 without PreAMP process then genomic contamination is still apparent For the optimal performance of RT FFPE PreAMP cDNA Synthesis procedures and best results from the PCR Array all RNA samples should be suspended in the RNase free water provided with the RT FFPE RNA Extraction Kit DO NOT use DEPC treated water 3 RNA Quality Control All RNA samples should also demo
26. nes are high this may suggest that either the total RNA input is less than the recommended lower limit of 100 ng or the quality of RNA is poor and affects the success of pre amplification Double check the concentration and quality of your RNA samples following the suggestions outlined in RNA Quality Control and Amount Considerations at the beginning of the protocol in this User Manual 2 Evidence of Genomic DNA Contamination You must perform either the recommended DNase treatment step included in the protocol of any RNA isolation kit you choose to use or treat your RNA sample after isolation with a good source of RNase free DNase such as TURBO DNA free kit from Ambion catalog number 1907 followed by re purification of the RNA You must also then use the RT First Strand cDNA Synthesis components in this kit with its genomic DNA elimination step If the genomic DNA contamination proves difficult to remove fold changes in gene expression may still be obtained However it will then be very important to validate any results for individual genes by a separate more rigorous real time PCR analysis that includes a minus RT control Apparent genomic DNA contamination may also indicate evidence of more general DNA contamination of other reagents tips and tubes See the Note about Preparing a Workspace Free of DNA Contamination at the beginning of the protocol in this User Manual 2 Evidence of Poor Reverse Transcription Efficiency If
27. nstrate consistent quality according to the following criteria a RNA Concentration and Purity by UV Spectrophotometry NOTE Prepare dilutions and measure absorbance in 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH i Azgo Az30 ratio should be greater than 1 7 A low 260 230 ratio indicates possible salt or guanidine contamination ii Azgo Azgo ratio should be from 1 8 to 2 2 A low 260 280 ratio indicates possible protein contamination iii Concentration by Azgo should be greater than 12 5 ng ul total RNA b Ribosomal RNA band integrity RNA from archived tissues samples is expected to show some degree of degradation Electrophorese a fraction of each RNA sample on a denaturing agarose gel or alternatively characterize the samples with a NanoChip 6000 on an Agilent BioAnalyzer Assess the extent of RNA degradation the usual length of FFPE RNA fragments is expected to be between 100 to 1000 bases Because some contaminants are difficult to detect by simply looking at RNA integrity and can be missed by UV spectrophotometry it is essential to choose the proper RNA isolation method for your biological sample as described above c The RT RNA QC PCR Array Optional The RT RNA QC PCR Array and the RT First Strand Kit each sold separately test for a number of RNA quality control parameters including e High and low housekeeping gene expression levels e Reverse transcription
28. onents and the RT PreAMP reagents in this kit have been optimized hand in hand for SYBR Green real time RT PCR detection further enhancing the sensitivity of our RT Profiler PCR Arrays The convenient and quick workflow of the RT FFPE PreAMP cDNA Synthesis Kit also makes this technology accessible for routine use in every research laboratory Version 1 0 Benefits of RT FFPE PreAMP cDNA Synthesis Kit Robust Performance on Partially Degraded Samples Demonstrated to provide high performance pre amplification without bias for FFPE samples Easy Workflow and Designed for Routine Use Simple and quick procedures with minimal hands on time to pre amplify target templates under 2 hours Enhanced Gene Profiling with Superior Sensitivity Increase the sensitivity of RT Profiler PCR Array by 2 order of magnitude to analyze compromised RNA FFPE RNA 100ng 11g GE gDNA Elimination Step 55 min BC3 i First Strand Synthesis P2 1 strand cDNA 1 4 of RT product i e 5uL RT PreAMP PCR Master Mix Specific for the RT Profiler PCR Step 8 Cycles Array of Your Choice Amplified template 20 min Side Reaction Reducer Tora NME eet sors CORE Real time PCR Detection of 89 Gene specific Amplification on RT Profiler PCR Array 2 5 3 hours RT Profiler PCR Arrays Figure 1 Workflow of RT PreAMP cDNA Synthesis Procedures for FFPE Samples Technical Support 888 503 3187 US 301 682 9200 D RT
29. ount that I can use with this kit This kit can be used with RNA input as little as 100 ng and as much as 1 yg of total RNA For successful results and maximum positive call rates we recommend that first time users try starting with 500 ng of total RNA For best results use a consistent amount of total RNA for all samples in a single experiment to be characterized and compared 3 Can I use the resulting amplified product from this kit for microarrays and GEArrays No The templates amplified from this kit are intended for analysis on individual RT Profiler PCR Arrays only 4 Can I use the resulting products amplified with one PreAMP Primer Mix for all PCR Arrays No Each RT FFPE PreAMP Primer Mix is specific to a catalogued RT Profiler PCR Array and can only amplify cDNA templates for the intended PCR Array To analyze on another PCR Array a separate PreAMP PCR reaction has to be performed using the correct RT FFPE PreAMP Primer Mix specific to the latter PCR Array If you have additional questions please check our website www SABiosciences com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Representatives at 1 888 503 3187 or 301 682 9200 Reference 1 von Ahlfen S Missel A Bendrat K Schlumpberger M Determinants of RNA Quality from FFPE Samples PLoS ONE 2007 Dec 5 2 12 e1261 Capsure and PicoPure are registered trademarks of Arcturus Agilent and BioAnalyzer are
30. re than two cycles between PCR Arrays being compared ii Larger differences in average C values between samples indicate the presence of different amounts of PCR amplification inhibitors in each sample and that all of the RNA Samples require further purification An average value of C that is consistently greater than 22 for all of your samples may indicate a problem with the cycling conditions or may simply be indicative of the relative sensitivity of your instrument See the Troubleshooting and FAQ section 3 Calculate the AC for each pathway focused gene iy each plate AG Ci GOI CG AVG NOTE Choosing the right normalization factor The expression level of the housekeeping genes chosen for normalization in the AAC method must not be influenced by your experimental conditions If one or more such genes have been previously identified by independent means and if the PCR Array reproduces those results use the average of their 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 Or if your RNA samples were of sufficiently high concentration for reliable quantification such that equal amounts of RNA from all samples were used simply use zero 0 in the place of the average of HK genes C for each group to be compared and rely on the consistency in the quantity and quality of your original input total RNA across your
31. redit card information Visa or MasterCard Shipping address Billing address For more information visit us at www SABiosciences com SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA Version 1 0 CONTENTS Background and Introduction 4 Il Materials Provided 6 Ill Additional Materials Required 7 IV Complementary Products 9 V Quick Protocol 10 VI Detailed Protocol 11 A RNA Preparation Quality and Amount Considerations 13 B First Strand cDNA Synthesis 16 C Pre Amplification of cDNA Target Templates 17 D Performing Real Time PCR with RT Profiler PCR Arrays 18 E Data Analysis 19 VII Alternative PreAMP Protocol for Individual RT qPCR Primer Assays 22 VIII Troubleshooting and Frequently Asked Questions 25 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of RT FFPE
32. registered trademarks of Agilent Inc LabChip is a registered trademark of Caliper Life Sciences NanoDrop is a registered trademark of NanoDrop Technologies StepOnePlus is a trademark of Applied Biosystems iCycler and MyiQ are registered trademarks of BioRad Laboratories Inc LabChip is a registered trademark of Caliper Life Sciences LightCycler is a registered trademark of Roche Applied Sciences SmartCycler is a registered trademark of Cepheid SYBR is a registered trademark of Molecular Probes TRIzol is a registered trademark of Invitrogen Mastercycler is a registered trademark of Eppendorf Technical Support 888 503 3187 US 301 682 9200 27 RT FFPE PreAMP cDNA Synthesis Kit RT FFPE PreAMP cDNA Synthesis Kit User Manual Part 1046A Version 1 0 3 24 2009 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O m 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com 28
33. s XpressRef Universal Total RNA o To control for DNA contamination introduced during reaction setup prepare a negative control reaction replacing template with water the so called no template control NTC o Tocontrol for genomic DNA contamination perform one assay for each gene of interest and each housekeeping gene using an equivalent volume of product from the No Reverse Transcription NRT reaction performed for each RNA sample d Experimental qPCR Cocktail Preparation per 25 ul reaction 2X SABiosciences RT qPCR SYBR Green 12 5 ul Master Mix DP RNase DNase free H20 10 5 ul RT qPCR Primer Assay 10uM 1 ul Diluted RT PreAMP PCR Reaction 1 ul Total Volume 25 ul 24 Version 1 0 Scale up the volume for each of the qPCR Cocktail reagents accordingly when multiple reactions are carried out to minimize pipetting variations e Load 25 ul of each of the above reaction to PCR tubes or a PCR plate appropriate for your real time thermal cycler f For the rest of qPCR assay protocol and data analysis please follow the instructions from Step C 2 to Step C 4 Real Time qPCR Primer Assay of the RT qPCR Primer Assays User Manual Technical Support 888 503 3187 US 301 682 9200 25 RT FFPE PreAMP cDNA Synthesis Kit VIII Troubleshooting and FAQs A Troubleshooting 1 High Ct Values for Many Genes including HouseKeeping Genes If the Ct values for all the housekeeping ge
34. tion Eppendorf Mastercycler ep realplex Instruments with ROX filter set Catalog Number Size PA 012 For 2RT Profiler PCR Arrays PA 012 12 For 12 RT Profiler PCR Arrays PA 012 24 For 24 RT Profiler PCR Arrays RT SYBR Green Fluorescein qPCR Master Mix Specifically designed for BioRad iCylcer MyiQ and iQ5 Instrumentation Catalog Number Size PA 011 For 2 RT Profiler PCR Arrays PA 011 12 For 12 RT Profiler PCR Arrays PA 011 24 For 24 RT Profiler PCR Arrays RT SYBR Green qPCR Master Mix Specifically designed for instrumentation that does not require a reference dye BioRad MJ Research Opticon Opticon 2 and Chromo 4 Roche LightCycler 480 System Eppendorf Mastercycler ep realplex Instruments without ROX filter set Catalog Number Size PA 010 For 2RT Profiler PCR Arrays PA 010 12 For 12 RT Profiler PCR Arrays PA 010 24 For 24 RT Profiler PCR Arrays 2 384 Well PCR Arrays Each 384 well PCR Array 4 pack Formats E amp G requires four 4 of the smaller size of the correct master mix for your instrument 4 X PA 01 Technical Support 888 503 3187 US 301 682 9200 g RT FFPE PreAMP cDNA Synthesis Kit E Equipments 1 A conventional programmable thermal cycler with 0 2mL tube heat block heated lid and 10 100 uL reaction capacity 2 For recommendations on specific real time instrumentation thermal cyclers with fluorescent detection see the list of master mixes and plate formats above NOTE The PCR
35. trand cDNA Synthesis components are also more compatible with our RT PreAMP PCR and RT qPCR master mixes than other enzymes or kits providing the PCR Arrays with maximum levels of sensitivity Version 1 0 NOTE Preparing a Workspace Free of DNA Contamination For accurate and reproducible PCR Array results it is very 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 The most common sources of DNA contamination are the products of previous experiments spread into the air of your working environment Please follow the recommendations below on how to set up and maintain a working environment free of DNA contamination 1 Wear gloves throughout the procedure Use only fresh PCR grade reagents H20 and lab ware tips and tubes 2 Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate your PCR workspace and lab ware pipettor barrels tube racks etc before each new use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 10 bleach to chemically inactivate and degrade any DNA 3 Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any lab ware tips or tubes containing PCR products or other DNA treat with 10 bleac
36. uccessful results and maximum positive call rates we recommend that first time users try starting with around 500 ng of total RNA It is also important to use a consistent amount of total RNA for all samples in a single experiment to be characterized and compared Version 1 0 B First Strand cDNA Synthesis NOTE The use of RT First Strand cDNA Synthesis Components in this kit is critical for successful pre amplification of templates for the RT Profiler PCR Arrays and detecting the Reverse Transcription Controls RTC in the PCR Arrays For more information on the importance of these components refer to the notes found on Pages 11 and 15 NOTE RNA samples must meet the standards of integrity and purity from protein organics and genomic DNA contamination described on the previous three pages 1 Clean up RNA by eliminating genomic DNA contamination a For each RNA sample combine the following in a sterile PCR tube Genomic DNA Elimination Mixture 1 reaction Total RNA 100 ng to 1 ug GE 5X gDNA Elimination Buffer 2 ul RNase free H20 Varied NOTE Use the similar amount of total RNA in this reaction for every sample First time users are recommended to start with 500 ng of total RNA Lower amounts of total RNA than 100 ng will dramatically affect the performance of the pre amplification process b Mix the contents gently with a pipettor followed by brief centrifugation c Incubate at 37 C for 15 min d Chill
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