RT Profiler PCR Array System
Contents
1. Technical Support support SABiosciences com www SABiosciences com 18 Version 3 5 Use Program 2 a three step cycling program for all other instruments For example the BioRad CFX96 BioRad Opticon Opticon 2 and Chromo 4 MJ Research Cycles Duration Temperature 1 10 minutes 95 C 15 seconds 95 C 40 30 to 40 seconds 55 C 30 seconds 72 C 1 The 10 minute step at 95 C is required to activate the HotStart DNA polymerase 2 Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle 3 Different instruments need different lengths of time to detect the fluorescent signal Choose the appropriate time for the annealing step 55 C for your instrument e Calculate the threshold cycle C for each well using the instrument s software i We highly recommend manually setting the Baseline and Threshold Values li To define the Baseline use the Linear View of the amplification plots and set the instrument to use the readings from cycle number two 2 through two 2 cycle values before the earliest visible amplification usually around cycle number ten 10 but no more than 15 iii To define the Threshold Value use the Log View of the amplification plots and place it above the background signal but within the lower one third to lower one half of the linear phase of the amplification plot iv IMPORTANT Ensure that the thresholds are the same across all PCR Array2. 15 16 97 18 19 20 24 22 23 24 o Bele tE a ead ka baa bod LE inal Laal SI LE Lea Ea vjolazzjoni t12 3 14 5 6 7 8 40 14 42 13 44 15 16 47 28 19 20 24 22 23 24 Sample 4 7412 3 4 5 6 718 10 14 12 13 14 15 16 97 18 19 20 24 22 23 24 VDIO Z SIF R O TIO mM M O a wm gt vjolazzjoni 12 3 EA 5 6 718 40 44 42 13 44 15 16 47 28 19 20 21 22 23 24 Figure 4 To load a 384 well format PCR Array add 10 ul of the Experimental Cocktail from each numbered sample into the staggered wells with the same number as indicated in the figure c Proceed to the next section Performing Real Time PCR Detection below Technical Support 888 503 3187 US 301 682 9200 17 RT Profiler PCR Arrays 4 Loading the PCR Array 384HT a CAREFULLY remove the PCR Array from its sealed bag b Add 10 ul of the 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 NOTE Change pipet tips following each addition to avoid any cross contamination between the wells or reactions 5 Performing Real Time PCR Detection NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument a CAREFULLY but t
3. Array Formats E or G NOTE Each 384 well plate characterizes four samples in separate sets of 96 wells staggered from one another by only one well The spacing between the tips of standard multi channel pipettors will allow you to properly skip rows or columns when adding each sample Be sure to load each sample into the correct set of wells Use Figure 4 as a guide a CAREFULLY remove the PCR Array from its sealed bag b Load sample cocktails to appropriate wells of the PCR Array preferably from a reservoir with an eight channel pipettor or a twelve channel pipettor but only using eight tips using the provided 384EZLoad Covers Catalog PA 384 and the figure below as a guide a Place Cover 1 white on the plate Add 10 uL of Sample 1 cocktail to the open wells Odd number wells of rows A C E G I K M amp O Remove amp discard the cover b Place Cover 2 yellow on the plate Add 10 uL of Sample 2 cocktail to the open wells Even number wells of rows A C E G I K M amp O Remove amp discard the cover c Place Cover 3 black on the plate Add 10 uL of Sample 3 cocktail to the open wells Odd number wells of rows B D F H J L N amp P Remove amp discard the cover d Place Cover 4 red on the plate Add 10 uL of Sample 4 cocktail to the open wells Even number wells of rows B D F H J L N amp P Remove amp discard the cover Sample 1 Sample 2 ARAKA EAKA NACAK 40 11 12 13 14
4. a spin column based clean up method such as SABiosciences s 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 this User Manual Also double check the quality of your RNA as described in Evidence of Poor Reverse Transcription Efficiency above Technical Support support SABiosciences com www SABiosciences com 24 Version 3 5 B Frequently Asked Questions 1 Will pipetting error affect the PCR Array results The passive reference dyes in the master mixes such as ROX and Fluorescein are used by the real time PCR systems to normalize variation from well to well Therefore these systems tolerate volume variations caused by pipetting error and evaporation 2 How can prevent the evaporation of reaction volume from the wells Be sure to carefully and completely seal the PCR Array with the optical thin wall 8 cap strips or the optical adhesive film before placing it in
5. runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays If the RNA sample quality has been adequately controlled the cycling program has been executed properly and the thresholds have been defined correctly then the value of CFPC should be 20 2 across all of your arrays or samples If not see the Troubleshooting and FAQ section v Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with our Data Analysis Template Excel file Technical Support 888 503 3187 US 301 682 9200 19 RT Profiler PCR Arrays 5 Recommended Quality Control Dissociation Melting Curve Run a melting curve program immediately after the above cycling program and generate a first derivative dissociation curve for each well in the entire plate using your instrument s software No more than one peak should appear in each reaction at temperatures greater than 80 C 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 ON If you decide not to obtain the dissociation curve immediately save the plates wrapped in aluminum foil at 20 C as is in case you need to perform this operation at a later point in time for troubleshooting purposes When ready simply warm the plate to room temperature place it into your
6. threshold cycle values of the control wells a Genomic DNA Control GDC i Calculate C O ii If the value is greater than 35 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 35 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 Kit s built in external RNA control also affect the reverse transcription of your messages of interest i Calculate AC AVG C AVG CPPS ii If this value is less than 5 then no inhibition is apparent iii If this value is greater than 5 then evidence of impurities that inhibited the reverse transcription phase of the procedure is evident See the Troubleshooting and FAQ section Technical Support 888 503 3187 US 301 682 9200 21 RT Profiler PCR Arrays 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 more 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 inhibit
7. G4 G5 G6 G10 G12 G G4 G5 G6 G10 G12 H G4 G5 G6 G10 G12 Figure 5 Layout of the Housekeeping Genes PCR Arrays D Data Analysis by the AC Method 1 For each sample average the duplicate determinations of the C values from each sample for each housekeeping gene Technical Support support SABiosciences com www SABiosciences com 26 Version 3 5 2 For each housekeeping gene calculate the AC or in other words the difference between the gene s C value in each experimental sample and the same gene s C value in the control sample 3 Choose the housekeeping genes with the smallest AC value across the samples of interest to normalize the results of your future RT PCR experiments for input total RNA loading More than one housekeeping gene may be chosen for your analyses Simply monitor the expression of all of these housekeeping genes and use their average C value as the normalization factor for each sample Technical Support 888 503 3187 US 301 682 9200 2 RT Profiler PCR Array User Manual Part 1022A Version 3 5 4 1 2009 fy SABiosciences Focus on Your Pathway 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com
8. NDATORY 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 Instrumentation 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 CFX96 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 3 PCR Array 384HT The PCR Array 384HT two 2 twelve 12 and twenty four 24 packs Formats E amp G require a quantity of tw
9. See Page 14 1 Experimental Cocktail Preparation Mix the following components in a 5 ml tube or a multi channel reservoir Plate Format 96 well 384 well 384HT Plate Format Designation A C D amp F E amp G E amp G 2X SABiosciences RT qPCR Master Mix 1275 ul 550 ul 2000 ul ee Strand cDNA Synthesis 102 ul 102 ul 102 ul ddH20 1173 ul 448 ul 1898 ul Total Volume 2550 ul 1100 ul 4000 ul NOTE This recipe provides an excess volume of ONLY 140 ul Very carefully add the cocktail to the PCR Array precisely as described below to insure that each well receives the required volume NOTE f you did not perform RNA quality control with a RT RNA QC PCR Array save the remaining 9 ul of the cDNA synthesis reaction at 20 C in case you need to perform one later for troubleshooting purposes 2 Loading the 96 Well PCR Array Formats A C D or F a CAREFULLY remove the PCR Array from its sealed bag b Add 25 ul of the 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 NOTE Change pipet tips following each addition to avoid any cross contamination between the wells or reactions c Skip the next page and proceed to Performing Real Time PCR Detection below Technical Support support SABiosciences com www SABiosciences com 16 Version 3 5 3 Loading the 384 Well PCR
10. and Kit 15 C Performing Real Time PCR 16 D Data Analysis 21 VI Troubleshooting and Frequently Asked Questions 24 Appendix Modified Protocol for Housekeeping Gene PCR Arrays 26 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 Profiler PCR Arrays 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 Patent pending RT Profiler PCR Arrays I Background and Introduction Real time reverse transcription RT PCR is the most sensitive and reliable meth
11. ciency of the RT First Strand Kit C 03 reaction with a primer set detecting the template synthesized from the kit s built in external RNA control The Positive PCR Control PPC tests the efficiency of the polymerase chain reaction itself using a pre dispensed artificial DNA sequence and the primer set that detects it The sets of replicate control wells GDC RTC and PPC also test for inter well intra plate consistency Custom PCR Arrays have your specified layout and the product information literature enclosed with the array reiterates that layout and the genes included Technical Support support SABiosciences com www SABiosciences com 6 Version 3 5 Il Materials Provided 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 Format For Real Time Instruments Plate All ABI standard blocks 7000 7300 7500 7700 7900 Bio Rad iCycler MyiQ iQ5 f A Chromo4 MJ Research PAVEN Eppendorf RealPlex Stratagene Mx3005p Mx3000p c ABI 7500 and 7900HT FAST 96 well blocks 96 well ABI StepOnePlus Bio Rad CFX 96 p Opticon and Opticon 2 MJ Research 7 OSEI Stratagene Mx4000 E ABI 7900HT 384 well block 384 well Roche LightCycler 480 96 well block 96 well G Roche LightCycler 480 384 well block 384 well NOTE The format of t
12. d reference dye ft cDNA gigi cDNA yy y PCRMix1 E PCRMix2 Sart Aliquot the Mixture Across Your PCR Arrays Each PCR Array profiles the expression of 84 pathway specific genes plus controls PCR Array 1 PCR Array 2 Perform Thermal Cycling Collect real time amplification data using your instrument s software Profile 1 Profile 2 10000 10000 1 LARS iail 1 RANA O 4 8 12 16 20 24 28 32 36 40 O 4 8 12 16 20 24 28 32 36 40 Analyze Changes in Gene Expression Analysis is straightforward A simple cut and paste operation puts the C values collected by your real time instrument into an analysis spreadsheet Fold changes in gene expression between your samples are calculated automatically Figure 1 Overview of the PCR Array Procedure Technical Support Version 3 5 2A Standard PCR Array Layouts 96 well PCR Array 30 minutes minutes aoe O 384 well PCR Array minutes 2 0 2 5 hours minutes O OQOOOHOOODDOOOOO D OQOODOOOOOOOOO POODOOOOOD OQOOOQOS Figure 2 Layout of the Cataloged Pathway Focused PCR Arrays 888 503 3187 US 301 682 9200 5 RT Profiler PCR Arrays Figure 2A Layout of the 96 Well and 384 Well PCR Arrays Wells A1 through G12 contain a real time PCR assay for genes from the same biological pathway or the same disease state or genes that are otherwise functionally related Wells H1 through H5 contain a housekeeping gene pa
13. e greater than 1 7 ii Azeo Azgo ratio should be greater than 2 0 iii Concentration by Azgo should be greater than 4 ug mI total RNA b Ribosomal RNA band integrity Electrophorese a fraction of each RNA sample on a denaturing agarose gel or on an Agilent BioAnalyzer using an RNA 6000 Nano LabChip and verify that there is a sharp distinction at the small side of both the 18S and 28S ribosomal RNA rRNA bands or peaks Any smearing or shoulder to the rRNA bands or peaks indicates that degradation has occurred in the RNA sample A B MW RNA FU MDA231 j 28S lt 18S NNW AUD wo uw gt n 1 1 20 25 30 35 40 45 50 55 s Figure 3 Good Ribosomal RNA Band Integrity Is Important for Optimal PCR Array Results Panel A displays an Agilent BioAnalyzer electropherogram of a high quality total RNA preparation showing sharp peaks without shoulders especially to the left of each peak for the 18S and 28S ribosomal RNA left to right Panel B right hand lane displays an analysis of the same high quality total RNA preparation by agarose gel electrophoresis demonstrating sharp bands especially at the bottom of each band for the 28S and 18S ribosomal RNA top to bottom 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 abo
14. e 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 bleach 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 11 RT Profiler PCR Arrays A RNA Preparation 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 consistent high quality RNA from every experimental sample Therefore the sample handling and RNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the RNA or decrease the effici
15. ency of if not block completely the enzyme activities necessary for optimal reverse transcription and real time PCR performance 1 Recommended RNA Preparation Methods High quality total RNA for your real time PCR experiment must be prepared using one of the following methods each specific for your biological sample a Cultured Cells Use SABiosciences 8 RT gPCR Grade RNA Isolation Kit Catalog PA 001 o the Qiagen RNeasy Mini Kit Catalog 74104 You must perform the recommended on column DNase treatment step b Tissue Samples First extract RNA from the tissue using the TRIzol protocol Invitrogen Catalog 15596 026 Be sure to use a sufficient amount of TRIzol reagent During homogenization add a volume of reagent at least ten times greater than the tissue volume Then after the ethanol precipitation step further clean up the RNA using SABiosciences S RT qPCR Grade RNA Isolation Kit Catalog PA 001 or the Qiagen RNeasy Mini Kit Catalog 74104 You must perform the recommended on column DNase treatment step c Whole Blood Samples Before RNA preparation red blood cells RBC must be removed from whole blood samples using a density gradient centrifugation medium for example Lymphoprep Greiner Bio One Catalog 1031966 The white blood cell fraction is then used for RNA isolation with SABiosciences S RT qPCR Grade RNA Isolation Kit Catalog PA 001 or the Qiagen RNeasy Mini Kit Catalo
16. fy SABiosciences User Manual RT Profiler PCR Array System Pathway Focused Gene Expression Profiling Using Real Time PCR See Purchaser Notification for limited use license and warranty information page 3 Part 1022A Version 3 5 4 1 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA RT ProfilerPCR Array System Pathway Focused Gene Expression Profiling Using Real Time PCR User Manual For Catalog Numbers Prefixed by PAHS PAMM and PARN Ordering and Technical Service Contact Information 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 credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www SABiosciences com SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA CONTENTS l Background and Introduction 4 Il Materials Provided 7 Ill Additional Materials Required 8 IV Complementary Products 9 V Protocol 10 A RNA Preparation and Quality Control 12 B RT First Str
17. g 74104 You must perform the recommended on column DNase treatment step Alternatively the PAXgene Blood RNA Kit Qiagen Catalog 762164 can also be used to prepare total RNA from whole blood samples d Total RNA Isolated Using a Phenol Based Method If you have already prepared total RNA from a any biological source material using a phenol based method such as TRIzol RNAzol etc you must clean up the RNA with SABiosciences s RT qPCR Grade RNA Isolation Kit Catalog PA 001 or the Qiagen RNeasy Mini Kit Catalog 74104 You must perform the recommended on column DNase treatment step e For Other Biological Samples Refer to the existing literature to find isolation protocols for high quality RNA from other biological samples or contact one of our Technical Support representatives Technical Support support SABiosciences com www SABiosciences com 12 Version 3 5 For best results from the PCR Array all RNA samples should be suspended in the RNase free water provided with the RNA Isolation kit DO NOT use DEPC treated water 2 RNA Quality Control For best results from the PCR Array all RNA samples should also demonstrate 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 b
18. h as 5 ug total RNA per array 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 use of the RT First Strand Kit C 03 maximizes the number of positive calls at low amounts 25 ng of total RNA over other sources of reverse transcriptase and first strand synthesis kits For successful results and maximum positive call rates we recommend that first time users try starting with anywhere from 0 5 ug to 1 0 ug 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 Technical Support support SABiosciences com www SABiosciences com 14 Version 3 5 B RT First Strand Kit C 03 NOTE The use of SABiosciences s RT First Strand Kit Cat No C 03 is critical for detecting the Reverse Transcription Controls RTC Wells H7 H9 and for obtaining the best results from the PCR Array For more information on the importance of this kit refer to the notes found on Pages 10 and 14 NOTE RNA samples must meet the standards of integrity and purity from
19. he 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 twelve 12 or twenty four 24 while the 384 well PCR Arrays Formats E and G are shipped in sets of four 4 The PCR Array 384HT is shipped in sets of two 2 twelve 12 or twenty four 24 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 Each 96x4 Format 384 Well PCR Array Formats E and G also includes one set of four 384EZLoad Covers Catalog PA 384 for each PCR Array provided in the package NOTE Each 384EZLoad Cover is for a Single Use ONLY Storage Conditions All components included in this kit are shipped at ambient temperature but must be stored at 20 C upon receipt When stored properly at 20 C their quality is guaranteed for 6 months Technical Support 888 503 3187 US 301 682 9200 7 RT Profiler PCR Arrays lil Additional Materials Required A RNA Isolation Kit See Page 12 for specific recommendations B RT First Strand Kit Cat No C 03 MANDATORY for a Complete and Successful Experiment For Reverse Transcription Control Detection RTC Wells H7 through H9 C SABiosciences RT qPCR Master Mix MA
20. her a 96 well or 384 well plate format containing either one or four replicates respectively of the 96 primer set panel See Figure 2 for the layout of typical PCR Arrays The PCR Array 384HT products catalog numbers greater than 3000 contain a 370 gene panel in a 384 well plate format SABiosciences s qPCR Assays Master Mixes and first strand kit have been optimized hand in hand for SYBR Green real time RT PCR detection providing the PCR Arrays with superior sensitivity and wide linear dynamic ranges The simplicity of the PCR Arrays also makes them accessible for routine use in every research laboratory Benefits of the RT Profiler PCR Arrays Pathway Focused Profile the expression of a panel of genes relevant to a pathway or disease state Simple and Accurate Simple real time PCR procedure provides high sensitivity and wide dynamic range Designed for Routine Use Bring expression profiling to almost any lab with a real time PCR instrument Combine microarray profiling capabilities with real time PCR performance Technical Support support SABiosciences com www SABiosciences com 4 How It Works Isolate RNA from your Experimental Samples Start with as little as 25 ng of total RNA 1 ug is recommended treat with DNase x Prepare cDNAs from your RNA Samples Use the RT First Strand Kit Cat No C 03 y cDNAT oDNA2 j Add cDNA to RT qPCR Master Mix RT qPCR Master Mixes contain SYBR Green an
21. ightly seal the PCR Array with the optical thin wall 8 cap strips Formats A and D or with the optical adhesive film Formats C E F and G NOTE Be sure that no bubbles appear in any of the wells of the PCR Array To remove bubbles tap the plate gently on the bench top or centrifuge the plate briefly b Place the plate on ice while setting up the PCR cycling program below c Place one plate in your real time thermal cycler If recommended by your instrument s user manual use a compression pad with the optical film sealed plate formats NOTE PCR Arrays containing experimental cocktail may be stored at 20 C wrapped in aluminum foil for up to one week until ready to run d Enter and run the appropriate program for your real time instrument below If prompted by your instrument 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 php Use Program 1 a two step cycling program for all of the following instrumentation All ABI Instruments 7000 7300 7500 7700 and 7900HT BioRad iCycler MyiQ cycler and iQ5 real time PCR detection systems All Stratagene Instruments Mx3000p Mx3005p and Mx4000p Eppendorf Mastercycler ep realplex and Roche LightCycler 480 Cycles Duration Temperature 1 10 minutes 95 C 40 15 seconds 95 C 1 minute 60 C
22. l Technical Support 888 503 3187 US 301 682 9200 23 RT Profiler PCR Arrays VI Troubleshooting and FAQs A Troubleshooting 1 Removal of Genomic DNA Contamination You must perform the on column DNase treatment step included in the protocol of SABiosciences s RT qPCR Grade RNA Isolation Kit PA 001 or Qiagen s RNeasy Mini Kit Catalog 74104 You must also then use SABiosciences s RT First Strand Kit C 03 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 The No Template Control NTC in the RT RNA QC PCR Array provides a sense of how well your technique minimizes the introduction of general DNA contamination into your assay system 2 Improving Poor Reverse Transcription Efficiency Double check the A260 A280 and A260 A230 ratios of your RNA samples and be sure to perform the dilutions for spectrophotometry in RNase free Tris pH 8 0 buffer If necessary re purify your RNA samples with
23. nel to normalize PCR Array data The product information included with each cataloged PCR Array contains a list of the pathway focused and housekeeping genes on the array Well H6 contains the Genomic DNA Control GDC Wells H7 through H9 contain replicate Reverse Transcription Controls RTC Wells H10 through H12 contain replicate Positive PCR Controls PPC The 384 well format of the PCR Arrays includes four replicates of the same 96 well format in which each two by two set of wells wells labeled 1 4 in gray above contains the same primer set represented by the 96 well designations Figure 2B Layout of the PCR Array 384HT Wells A1 through P10 1 370 each contain a real time PCR assay for genes from the same biological pathway or the same disease state or genes that are otherwise functionally related Wells P12 through P15 contain a housekeeping gene panel to normalize PCR Array data The product information included with each cataloged PCR Array contains a list of the pathway focused and housekeeping genes on the array Wells P16 through P18 contain replicate Genomic DNA Controls GDC Wells P19 through P21 contain replicate Reverse Transcription Controls RTC Wells P22 through P24 contain replicate Positive PCR Controls PPC The Genomic DNA Control GDC is an assay that specifically detects non transcribed genomic DNA contamination with a high level of sensitivity The Reverse Transcription Control RTC tests the effi
24. o 2 of the correct master mixes for your instrument of the size for the corresponding 96 well PCR Array packs 2 X PA 01 or 2 X PA 01 12 or 2 X PA 01 24 Technical Support support SABiosciences com www SABiosciences com 8 Version 3 5 D Equipment 1 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 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 2 Calibrated Multi Channel Pipettor 3 RNase DNase free pipette tips and tubes IV Complementary Products A RT RNA QC PCR Array Optional Pick the correct catalog number for your species of interest See below and the correct plate format for the instrument in your lab See table on Page 7 Human RT RNA QC PCR Array Cat No PAHS 999 Mouse RT RNA QC PCR Array Cat No PAMM 999 Rat RT RNA QC PCR Array Cat No PARN 999 B 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 Technical Support 888 503 3187 US 301 682 9200 9 RT Profiler PCR Arrays V P
25. od for gene expression analysis Its wide dynamic range makes real time RT PCR the preferred choice for the simultaneous quantification of both rare and abundant genes in the same sample The RT Profiler PCR Array takes advantage of real time PCR performance and combines it with the ability of microarrays to detect the expression of many genes simultaneously RT Profiler PCR Arrays are designed to analyze a panel of genes related to a disease state or biological pathway The product is especially suitable for researchers who are more familiar with or prefer real time PCR technology but are looking for the multi gene profiling capabilities of a microarray To complete the PCR Array procedure start by converting your experimental RNA samples into first strand cDNA the template for the polymerase chain reaction using our RT First Strand Kit See Figure 1 for an overview of the PCR Array procedure Then mix your template with one of our instrument specific and ready to use RT qPCR 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 Each array contains a panel of 96 or 384 primer sets for a thoroughly researched set of 84 or 370 relevant pathway or disease focused genes plus five housekeeping genes and three RNA and PCR quality controls The PCR Arrays are available in eit
26. oducts that would otherwise cause false positive signals The Reverse Transcription Controls RTC on the PCR Array can only be 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 transcriptases or first strand synthesis kits The buffer components and the magnesium concentration in our RT First Strand Kit are also more compatible with our PCR master mixes than other enzymes or kits providing the PCR Arrays with maximum levels of sensitivity with ng to ug amounts of total RNA Technical Support support SABiosciences com www SABiosciences com 10 Version 3 5 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 Decontaminat
27. ors 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 in each plate AGs ceo CAS HKG 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 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 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 gr
28. oup 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 support SABiosciences com www SABiosciences com 22 Version 3 5 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 L 2 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 Gal Te E g C G0 CHKG I _ oat geike E 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 9 Atrlexpt a Acieontro 244 Where AAC is equal to AC expt AC control The complete calculation is as follows 9 ACHHKE expt E g ICdG01 CAHKG expt 7 gat expt S g t yael control 2 MPAP CUHK Cn tro 2 control 9 ACAHKG contro
29. pheid SYBR is a registered trademark of Molecular Probes TRIzol is a registered trademark of Invitrogen Mastercycler is a registered trademark of Eppendorf Mx3000P Mx3005P and Mx4000 are registered trademarks of Stratagene Technical Support 888 503 3187 US 301 682 9200 25 RT Profiler PCR Arrays Appendix Modified Protocol for Housekeeping Gene PCR Arrays B First Strand cDNA Synthesis Perform a first strand cDNA synthesis reaction for each sample to be characterized on the array including one sample representing your biological or experimental control C Perform Real Time PCR 1 Experimental Cocktail Preparation Mix the following components in a 1 ml tube or a multi channel reservoir 2X SABiosciences RT qPCR Master Mix 337 5 ul Diluted first strand cDNA synthesis reaction 27 ul ddH2O0 310 5 ul Total volume 675 ul 2 Adding samples to PCR Array NOTE Organize your sample loading onto the arrays very carefully making sure to characterize each sample in duplicate and to include a replicate of the control sample on each plate For example up to four samples can be characterized in duplicate on a single array or duplicate determinations may be made on two separate arrays for larger numbers of samples Housekeeping Genes 4 5 6 10 12 A G4 G5 G6 G10 G12 B G4 G5 G6 G10 G12 Bile G4 G5 G6 G10 G12 S D G4 G5 G6 G10 G12 BE G4 G5 G6 G10 G12 F
30. protein organics and genomic DNA contamination described on the previous two pages 1 Genomic DNA Elimination Mixture a For each RNA sample combine the following in a sterile PCR tube Total RNA 25 0 ng to 5 0 ug GE 5X gDNA Elimination Buffer 2 0 RNase free H20 to a final volume of 10 0 ul NOTE Use the same amount of total RNA in this reaction for every sample First time users are recommended to start with 0 5 or 1 0 ug of total RNA for 96 well plate formats or with 0 2 to 0 5 ug of total RNA for 384 well plate formats Lower amounts of total RNA than 100 ng will dramatically increase the false negative rate of the PCR Array method b Mix the contents gently with a pipettor followed by brief centrifugation c Incubate at 42 C for 5 min d Chill on ice immediately for at least one minute 2 Prepare the RT Cocktail RT Cocktail 1 reaction 2 reactions 4 reactions BC3 5X RT Buffer 3 4ul 8 ul 16 ul P2 Primer amp External Control Mix 1 ul 2 ul 4 ul RE3 RT Enzyme Mix 3 2 ul 4 ul 8 ul RNase free H20 3 ul 6 ul 12 ul 3 First Strand cDNA Synthesis Reaction Add 10 wl of RT Cocktail to each 10 ul Genomic DNA Elimination Mixture Mix well but gently with a pipettor c Incubate at 42 C for exactly 15 min and then immediately stop the reaction by heating at 95 C for 5 minutes d Add 91 ul of ddH20 to each 20 ul of cDNA synthesis reaction Mix well e Hold the finished First S
31. real time instrument and run the melting program described above NOTE Be sure to visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed make a note of which wells so that you may qualify your data analysis appropriately NOTE 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 See also the Note on Preparing a Workspace Free of DNA Contamination Technical Support support SABiosciences com www SABiosciences com 20 Version 3 5 D Data Analysis AAC Method NOTE PCR Array Data Analysis Web Portal Access our free PCR Array Data Analysis Web Portal from the following address http www SABiosciences com pcrarraydataanalysis php 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 Change all C values reported as greater than 35 or as N A not detected to 35 At this point any C value equal to 35 is considered a negative call 2 Examine the
32. rotocol 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 NOTE Master Mix and First Strand Synthesis Considerations The performance of our RT Profiler PCR Arrays is only guaranteed with SABiosciences RT2 qPCR Master Mixes and the RT First Strand Kit Therefore the use of the complete RT Profiler PCR Array System is absolutely essential for obtaining accurate real time PCR profiling results 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 The RT First Strand Kit includes a proprietary buffer to eliminate any residual genomic DNA contamination in your RNA samples before it can be amplified into secondary pr
33. to your thermal cycler Also be sure to use a compression pad with the plate formats using the optical film seal Formats C E F and G as directed by the manufacturer of your real time PCR instrument 3 How reliable are the results from the RT Profiler PCR Array Assuming the use of good consistent experimental technique real time PCR methods such as the PCR Array provide very reproducible results To insure the reliability of your results and to reliably detect smaller fold changes in gene expression from the PCR Array the performance of replicate determinations such as biological triplicates is highly recommended The Data Analysis Template available from our website for the PCR Array uses your replicate PCR Array data to calculate t test p values and to generate a Volcano Plot illustrating the statistically significant fold changes in gene expression 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 ABI ROX and StepOnePlus are registered trademarks of Applera Corporation Opticon 2 Chromo4 iQ5 iCycler CFX96 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 Ce
34. trand cDNA Synthesis Reaction on ice until the next step or store overnight at 20 C 4 RNA Quality Control Check Optional If desired proceed to characterize a small aliquot 6 ul of the diluted cDNA template on the correct species specific and instrument specific RT RNA QC PCR Array following the instructions provided in its User Manual Save the remainder at 20 C E Technical Support 888 503 3187 US 301 682 9200 15 RT Profiler PCR Arrays C Performing Real Time PCR NOTE The use of SABiosciences s 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 Pages 8 and 10 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 NOTE f unsure of your RNA quality or isolation technique examine the quality of your RNA before this step using SABiosciences s species and instrument specific RT RNA QC PCR Arrays
35. ve Technical Support 888 503 3187 US 301 682 9200 13 RT Profiler PCR Arrays 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 and polymerase chain reaction efficiency e Genomic and general DNA contamination 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 3 Genomic DNA Contamination Eliminating genomic DNA contamination is essential for obtaining optimal real time gene expression profiling results using the PCR Array The Genomic DNA Control in each PCR Array specifically tests for genomic DNA contamination in each sample during each run A GDC threshold cycle value less than 35 indicates the presence of a detectable amount of genomic DNA contamination that should be addressed We highly recommend performing the on column DNase treatment step in the RT qPCR Grade RNA Isolation Kit PA 001 or the Qiagen RNeasy Mini Kit Catalog 74104 followed by using the RT First Strand Kit C 03 to remove any and all residual contamination from your RNA samples 4 Amount Considerations The PCR Array System yields results with as little as 25 ng or as muc
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