User Manual RT Profiler™ PCR Array
Contents
1. Specifically designed for instrumentation not requiring a reference dye D Equipment Real time instrumentation thermal cycler with fluorescent detection For instrument recommendations see the list of master mixes above NOTE The PCR Arrays are NOT recommended for the Cepheid SmartCycler or the Roche LightCycler amp 2 0 due to the different non traditional hot block arrangements in those instruments 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 005 Mouse XpressRef Universal Total RNA Cat No GA 006 Rat XpressRef Universal Total RNA Cat No GA 007 Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 ss V 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 NOTE Master Mix Considerations The use of SuperArray s RT Real Time SYBR Green PCR master mixes is absolutely essential for obtaining accurate results with the RT Profiler PCR Arrays The chemically modified and tightly controlled HotStart enzyme in these master mixes uniquely provides more accurate SYBR Green results by preventing the ampl2. total RNA from any biological source material using a phenol based method such as TRIzol RNAzol etc you must clean up the RNA with the Qiagen RNeasy Mini Kit Catalog 74103 to insure optimal performance Be sure to include the recommended DNase treatment step For Other Biological Samples Refer to existing literature to find isolation protocols for high quality RNA from other biological samples or contact a Technical Support representative For best results from the PCR Array all RNA samples should be suspended in the RNase free water provided with the RNA Isolation kit not DEPC treated water or alternatively in RNase free 10 mM Tris buffer pH 8 0 Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 10 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 an RNase free 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH i Concentration by Azgo should be greater than 4 ug ml total RNA ii A260 A280 ratio should be greater than 2 0 iii Azgo A230 ratio should be greater than 1 7 b Ribosomal RNA band integrity Electrophorese a fraction of each RNA sample on a denaturi
3. detect the fluorescent signal Choose the annealing step 55 C time appropriate for your instrument c Calculate the threshold cycle C for each well using the instrument s software NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 15 4 Optional Quality Control a Dissociation Melting Curve Run a melting curve program immediately after the above PCR 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 sec OPTICS ON If you decide not to obtain the dissociation curve immediately save the plates 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 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
4. for more information 2 Change all C values reported as greater than 35 or as N A not detected to 35 Any C value equal to 35 is considered a negative call 3 When biological and or technical replicates are performed calculate the average C value of each gene each well across those replicate arrays for each treatment group 4 Calculate the AC for each pathway focused gene in each treatment group AC group 1 average C average of HK genes C for group 1 array AC group 2 average C average of HK genes C for group 2 array NOTE 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 Ci value of all five housekeeping genes but only if the difference in the average values between the two groups to be compared is less than one 1 cycle Otherwise 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 5 Calculate the AAC for each gene across two PCR Arrays or groups
5. is as follows Ct GOI expt G HKG exot C GOI C HK expt 2 C HKG expt f 2 2 AC expt MAG 5 Ct GOI control 7 5 Ci GOI C HK control p 5 AC control p C HKG control An Excel file downloadable from our web site automatically performs these calculations upon including a gene list and threshold cycle data from a real time instrument This Data Analysis template presents the results in a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included To download the template visit the PCR Array home page at the following web address http www superarray com PCRArrayPlate php Click the PCR Array Data Analysis link found in the lower Product Support section of the gray right hand sidebar Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 18 E Alternate Protocol End Point PCR Detection amp Data Analysis Method Additional Materials Required 1 RNA Isolation Kit See real time protocol for specific suggestions 2 ReactionReady First Strand cDNA Synthesis Kit Cat No C 01 3 ReactionReady HotStart Sweet PCR Master Mix Cat No PA 007 4 SYBR Green I Molecular Probes Cat No 87563 10 000X in DMSO 5 Equipment Standard thermal cycler and 96 well microplate fluorescence reader and appropriate optical 96 well plates
6. is observed make a note of which wells so that you may qualify your data analysis appropriately b Agarose gel electrophoresis In either case save the completed reactions at 20 C in case agarose gel electrophoresis characterization is also needed for troubleshooting purposes No more than one band should be apparent in each lane i Mix 10 ul of each reaction with 2 ul of 6X agarose gel loading buffer ii Load each sample into separate wells of a 2 agarose gel containing 0 5 ug ml ethidium bromide in 1X TAE iii Load an appropriate amount of 100 bp DNA Step Ladder Promega G695A in an adjacent lane iv Electrophorese in 1X TAE at 90V for 40 minutes or before the tracking dye runs off the gel v Capture an image of the gel with a UV Trans Illuminator using a Gel Doc Station CCD camera or high speed film Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 16 D Data Analysis AAC Method 1 Examine the threshold cycle of the negative controls The No Template Control NTC tests for DNA contamination in your PCR system while the No Reverse Transcription NRT control tests for contamination of the original RNA with genomic DNA Both threshold cycles should be greater than 35 If the threshold cycle for either of these controls is less than 35 then the presence of DNA contamination is evident See the Troubleshooting Guide
7. lab with a real time PCR instrument Combine microarray profiling capabilities with real time PCR performance Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 5 Figure 1 Overview of the PCR Array procedure How It Works Prepare cDNAs from your RNA samples Use a ReactionReady First Strand cDNA Synthesis Kit Cat No C 01 F i cDNA1 5 cDNA 2 Add cDNA Directly to RT PCR Master Mix Master Mixes contain SYBR Green and Reference Dyes cDNA bd cDNA y PCR Mix 1 SS PCRMix2 SES Aliquot the Mixture Across Your PCR Arrays Each PCR Array Profiles the Expresion of 84 Pathway Specific Genes plus Controls PCR Array 1 PCR Array 2 x Perform Thermal Cycling Then Determine Fold Changes in Expression by AAC Method Profile 1 Profile 2 Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 e 1 2 3 4 5 6 7 8 9 10 11 12 Figure 2 Layout of the Cataloged PCR Arrays r n M U O U Ft Wells A1 through G12 contain primers for genes from the same biological pathway G1 G84 The product information included with each array contains a list of these genes You will add aliquots of the same cDNA template to each of these wells Wells H1 through H5 contain a panel of housekeeping genes HK1 HK5 used for normal
8. mouse Me tlsofieepina PCR Mas House ids E 1 2 3 4 10 11 12 so duies G9 G10 crommuootusc D Data Analysis by the AAC Method 1 For each sample average the duplicate determinations of the C values from each sample for each housekeeping gene 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 Ci 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 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 23 This page is intentionally left blank Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 RT Profiler PCR Array 4 21 2006 24 e PCR BASED PATHWAY FOCUSED GENE EXPRESSION PROFILING IN A 96 WELL FORMAT BIOMOL GmbH Waidmannstr 35 22769 Hamburg info biomol de www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800
9. optimal real time gene expression profiling results using the PCR Array The no reverse transcription NRT control in well H11 specifically tests for genomic DNA contamination and should yield real time C values greater than 35 If the C values are less than 35 then genomic contamination is apparent While isolating your total RNA be sure to include any DNase treatment steps in the recommended RNA isolation procedure You may also re treat your RNA sample after isolation with a good source of RNase free DNase followed by re purification using a spin column based method e g the Qiagen RNeasy Mini Kit Increase the number of units of enzyme and incubation time beyond the recommendations made by the original manufacturer for the RNase free DNase 4 Amount Considerations The PCR Array will yield relative gene expression profiles with as little as 50 ng or as much as 5 ug total RNA per array However the use of smaller amounts of RNA will likely not detect genes expressed at a low level In other words the optimal amount of starting material depends upon 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 For successful results
10. per array C format PCR Arrays are shipped in sets of two or twelve 2 or 12 and come in three different plate formats each tailored to a specific subset of real time PCR instruments Format For Real Time Instruments ABI 7000 7300 7500 standard 7700 7900 standard A Bio Rad iCycler MyiQ Bio Rad MJ Research Chromo 4 Stratagene Mx3005p Mx3000p Mx4000 ABI 7500 FAST 7900 FAST Bio Rad MJ Research Opticon 2 and 4 UO OU The format of the PCR Array is indicated by the last digit of the catalog number NOTE Be sure that you have the correct PCR Array format for your instrument before starting the experiment Storage Conditions All components included in this kit are shipped at ambient temperature but must be stored at 20 C where they are guaranteed as is for 6 months from the date received Ill Additional Materials A RNA Isolation Kit See Page 8 for specific suggestions B ReactionReady First Strand cDNA Synthesis Kit Cat No C 01 C SuperArray RT Real Time SYBR Green PCR Master Mix MANDATORY for a Complete and Successful Experiment Be sure to pick the correct one for the instrumentation in your laboratory RT Real Time SYBR Green ROX Cat No PA 012 Specifically designed for all ABI and Stratagene Instrumentation RT Real Time SYBR Green Fluorescein Cat No PA 011 Specifically designed for BioRad iCylcer and MyiQ RT Real Time SYBR Green Cat No PA 010
11. 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 SuperArray Bioscience 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 Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 e I BACKGROUND AND INTRODUCTION Real time reverse transcription RT PCR is the most sensitive and reliable method for gene expression analysis Its wide dynamic range makes real time RT PCR the preferred choice for the simultaneous quantification of 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 capabili
12. 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 Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 21 6 How should I define the fluorescence intensity baseline for the calculation of the threshold cycle values Usually the software that accompanies your real time PCR system automatically calculates the average fluorescence background and the threshold cycle C values for each well in the array plate Sometimes the expression level of the 18S rRNA housekeeping gene is high enough to influence the automatic calculation of the baseline or threshold used to determine the C values from the rest of the wells As a result the software may report the 18S rRNA as not detectable and or may artificially inflate the C values of the rest of the genes in the array In these situations manually setting the baseline or threshold position for the C value calculations will be necessary by following the recommended procedure below a Select every well except the 18S rRNA well H1 for data analysis b Choose the first few cycles for example cycles 2 through 10 as those representing the average background fluorescence to help define the threshold value Alternatively you may define your own fluorescence threshold level
13. we recommend starting with as much input material as possible for example 0 5 or 1 0 ug of total RNA Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 12 B First Strand cDNA Synthesis Using SuperArray Catalog Number C 01 sold separately NOTE The buffer components and the magnesium concentration of the reverse transcription reaction using this kit are more compatible with our PCR master mixes than other sources of first strand cDNA synthesis kits or reverse transcriptases 1 Prepare the Annealing Mixture For each RNA sample combine the following in a sterile PCR tube Total RNA 0 05 to 5 0 ug Buffer P 1 0 ul RNase free H20 to a final volume of 10 0 ul Use the same amount of total RNA in this reaction for every sample Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a thermal cycler at 70 C for 3 min Cool to 37 C and incubate there for 10 min NOTE First time users are recommended to start with 0 5 or 1 0 ug of total RNA NOTE Be sure to save at least 1 ul of each RNA preparation to set up your no reverse transcription NRT controls in well H11 of each PCR Array 2 Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is incubating at 37 C RT Cocktail 1 reaction 2 reactions 4 reactions Buffer BC 5X RT Buffer 4 u
14. 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 ss CONTENTS Background and Introduction 4 Il Kit Contents Materials Provided 7 IIl Additional Materials Required T IV Complementary Products 7 V Protocol A RNA Preparation and Quality Control 9 B First Strand cDNA Synthesis 12 C Performing Real Time PCR 13 D Data Analysis 16 E Alternate Protocol End Point PCR Detection amp Data Analysis Method 18 VI Troubleshooting and Frequently Asked Questions 20 Appendix Modified Protocol for Housekeeping Gene PCR Arrays 22 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 SuperArray Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SuperArray Bioscience 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 Array includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to
15. 2466652 D 0800 2466651 cc sess co 99 so sess O biomol eo oo oo eo oo oo ee oo ee eoe eo oo eo oo oo eo eo oo 9 99 99 99 cooo se Technical Support 49 40 853260 23 27 37 ts biomol de www SuperArray com
16. A SuperArray Bioscience Corporation User Manual Part 1017A Version 1 5 4 21 2006 BIOMOL GmbH Waidmannstr 35 O 22769 Hamburg b info biomol de iomo www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D RT Profiler PCR Array PCR BASED PATHWAY FOCUSED GENE EXPRESSION PROFILING IN A 96 WELL FORMAT See Purchaser Notification for limited use license and warranty information page 3 RT Profiler PCR Array User Manual Version 1 5 4 21 2006 2 RT Profiler PCR Array For Catalog Numbers Prefixes APH APM and APR PCR Based Pathway Focused Gene Expression Profiling in a 96 Well Format USER MANUAL ORDERING INFORMATION AND TECHNICAL SERVICE e TEL 0800 2466651 D 49 8532600 outside D e FAX 0800 2466652 D 49 85326022 outside D e ON LINE ORDER www biomol de e E MAIL info biomol de to place an order ts biomol de for technical support You may place orders by phone 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 http www superarray com or http Awww biomol de SuperArray Bioscience Corp 7320 Executive Way Suite 101 Frederick MD 21704 USA Technical Support 0800
17. 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 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 17 NOTE Detailed Mathematical Explanation of AAC Data Analysis Method Due to the inverse proportional relationship between threshold cycle and the original gene expression level and the doubling of the amount of product of every cycle the original expression level L for each gene of interest is expressed as Ci 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 C GOI i Cx GOI C HK _ ACi C HKG 7 To determine fold change in gene expression the normalized expression of the GOI in the experimental sample is divided by the normalized expression of the same GOI in the control sample 2 AC expt ANE AC control i ntrol F AC control Where AAC is equal to AC expt AC contro The complete calculation
18. c Click the appropriate recalculate or analysis button according to the instrument manufacturer instructions and the software will recalculate and re plot all of the amplification curves d Examine the semi log amplification curves cycle vs Log intensity Be sure that the threshold value crosses each of these curves within the lower two thirds of exponential phase but above any background noise within the assay The lower the threshold value the more sensitive the results will be NOTE f you defined the threshold level yourself you will also need to use the same threshold for all samples arrays that you plan to compare to one another You may need to repeat this process a few times for each sample in order to meet these criteria e Export these C values f Select only the 18S rRNA well H1 for data analysis In this case define your own fluorescence threshold level close to that defined for the other wells above instead of relying on an automatic determination as described above Repeat steps c through e and export this C value separately g Continue with the data analysis and calculation of fold changes in gene expression as described in this User Manual If you have additional questions please check our website www superarray com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Representatives at 0800 2466651 or 49 40 853260 23 27 37 RNeasyO is a registered trad
19. designed for your reader Protocol 1 Perform Sample Preparation and Addition of Samples to PCR Array as described for real time PCR 2 Also prepare a 5X solution of SYBR Green Add 50 ul 10 000X concentrate SYBR Green to 5 ml DMSO Mix well Aliquot and store at 20 C On the day of the experiment generate a 5X SYBR Green Solution For each plate mix 0 5 ml of the 100X SYBR Green Stock with 9 5 ml ddH2O Store on ice Use only on the same day Discard any remainder IMPORTANT PROTECT SYBR Green SOLUTIONS FROM LIGHT NOTE To minimize variation always freshly prepare and use the same batch of 5X SYBR Green for all arrays in the same experiment Scale up the recipe as needed 3 Place one plate in thermal cycler Enter and run the following program Cycles Duration Temperature 1 10 minutes 95 C 35 30 seconds 95 C 30 seconds 55 C 30 seconds 72 C The 10 minute step at 95 C is required to activate the HotStart DNA polymerase 4 Quantify the PCR yield from each well at the end of the PCR program a Transfer 3 ul of the PCR from each well to a fresh 96 well plate not provided b Add 100 ul of 5X SYBR Green to each well in the new plates Change the pipet tips each time to avoid cross contaminating the reactions c Place the new plate in a 96 well microplate fluorescence reader d Read the fluorescence intensity with excitation at 485 nm and emission at 530 nm T
20. e calibrated before beginning this procedure Also be sure to not introduce any bubbles into the wells of the PCR Array 1 Sample Preparation a For wells A1 through H5 Mix the following components in a 5 ml tube or a multi channel pipettor reservoir 2X SuperArray PCR master mix 1225 ul Diluted first strand cDNA synthesis reaction 98 ul ddH O 1127 ul Total volume 2450 ul b For wells H6 through H10 In separate tubes prepare five 5 10 fold serial dilutions of the cocktail generated above for wells A1 H5 For WELL H6 H7 H8 H9 H10 1X PCR master mix 27u 27 nll 27 nll 27u 27 ui Material used for previous well 3 ul 3u 3u 3y 3ul c In another separate tube mix 1 ul of a 1 100 dilution of your original input total RNA with 24 ul of 1X PCR master mix to use as the no reverse transcription NRT control in well H11 d In another separate tube prepare a 25 ul aliquot of 1X PCR master mix to use as the no template control NTC in well H12 To prepare 1X PCR master mix combine 100 ul each 2X SuperArray PCR master mix and ddH20 More specifically mix 3 ul of the cocktail generated for wells A1 through H5 with 27 ul of 1X PCR master mix Generate four more 10 fold serial dilutions of this mixture into 1X PCR master mix Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Vers
21. emark of Qiagen SYBR is a registered trademark of Molecular Probes iCycler amp and MyiQ are registered trademarks of BioRad Laboratories Inc SmartCycler is a registered trademark of Cepheid LightCycler is a registered trademark of Roche Applied Sciences Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 22 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 experimental control C Perform Real Time PCR 1 Sample Preparation a To characterize each sample in duplicate Mix the following components in a 1 ml tube or a multi channel pipettor reservoir 2X SuperArray PCR master mix 337 5 ul Diluted first strand cDNA synthesis reaction 27 ul ddH2O 310 5 ul Total volume 675 ul Skip steps C 1 b C 1 c and C 1 d 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 Figure 4 Layout of the
22. he gain may need to be adjusted to place the whole plate in a detectable range Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 19 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 End Point PCR Data Analysis 1 Calculate the background corrected relative expression level for each gene Background correction removes the contribution of non specific signal intensity from each gene specific fluorescence intensity value Subtract the fluorescence reading in each well by the fluorescence reading of the No Template Control NTC Well H12 Change any resulting negative values to zero 2 Determine which housekeeping gene s to use for normalization Normalization removes the influence of systematic variation between arrays so that numbers can be compared between them Only use housekeeping genes wells H1 through H5 that do not drastically change their values between the experimental conditions to be compared For each PCR Array average all of the background corrected fluorescence intensity values for all useful housekeeping genes on that array 3 Normalize the results Divide the background corrected fluorescence intensity value of each gene of interest on each ar
23. ification of primer dimers and other non specific products The RT Real Time PCR Master Mix formulations also include other proprietary chemical components that significantly minimize primer dimer formation ensuring high amplification efficiencies for even the most difficult to amplify genes The performance of our RT Profiler PCR Arrays is only guaranteed with our RT Real Time SYBR Green PCR master mixes and not any other source of master mix In fact when we test other sources of master mix with our primer sets 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 are using the correct master mix for the instrumentation in your laboratory 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 reagent
24. ion 1 5 4 21 2006 14 2 Adding samples to PCR Array a CAREFULLY remove the plate seal from the PCR Array b Add 25 ul of the appropriate cocktail to each well of the PCR array Change the pipet tips each time to avoid cross contaminating the reactions c CAREFULLY but tightly seal the PCR Array with the optical thin wall 8 cap strips Formats A B and D or with the optical adhesive film Format C 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 if possible centrifuge the plate briefly d Place the plate on ice while setting up the appropriate PCR program below 3 Performing Real Time PCR Detection a Place one plate in your real time thermal cycler b Enter and run the appropriate program for your real time instrument For the ABI Instrumentation 7000 7300 7500 and 7900 Cycles Duration Temperature 1 10 minutes 95 C 40 15 seconds 95 C 1 minute 60 C For the BioRad iCycler amp and all other instrumentation Cycles Duration Temperature 1 10 minutes 95 C 40 15 seconds 95 C 30 to 40 seconds 55 C 30 seconds 72 C The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle Different instruments need different lengths of time to
25. ivities 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 Cultured Cells Use the Qiagen RNeasy Mini Kit Catalog 74103 Be sure to include the recommended DNase treatment step Tissue Samples Use a two step protocol First extract RNA from the tissue using the TRIzol amp 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 the Qiagen RNeasy Mini Kit Catalog 74103 Be sure to include the recommended DNase treatment step 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 the Qiagen RNeasy Mini Kit Catalog 74103 Be sure to include the recommended DNase treatment step Alternatively the PAXgene Blood RNA Kit Qiagen Catalog 762134 can also be used to prepare total RNA from whole blood samples Total RNA Isolated Using a Phenol Based Method If you have already prepared
26. izing the PCR Array data Again refer to your array s product information for the list of the housekeeping genes You will also add aliquots of the same cDNA template used for wells A1 through G12 to each of these wells Wells H6 through H10 contain the same housekeeping gene primers as well H5 HK5 These wells are designed to estimate the linear dynamic range of the assay You will add a different 10 fold serial dilution the cDNA template to each of these wells Well H11 also contains the same housekeeping gene primers as well H5 HK5 This reaction will contain your RNA diluted into master mix and will serve as the no reverse transcription control NRT to estimate the level of genomic DNA contamination in the RNA sample Well H12 also contains the same housekeeping gene primers as well H5 HK5 This reaction will contain only master mix and will serve as the no PCR template control NTC or water control to estimate the level of overall DNA contamination in the PCR system Custom PCR Arrays have your specified layout and the product information enclosed with the array specifies the layout and the genes included Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 7 Il Materials Provided Each PCR Array includes the array itself and either twelve 12 optical thin wall 8 cap strips A and B formats or one 1 optical adhesive film
27. l 8 ul 16 ul RNase free H20 4 ul 8 ul 16 ul RI RNase Inhibitor 1 ul 2 ul 4 ul RE Reverse Transcriptase 1 ul 2 yu 4 ul Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step 3 First Strand cDNA Synthesis Reaction Add 10 ul of RT Cocktail to each 10 ul Annealing Mixture Mix well but gently with a pipettor and continue incubation at 37 C for 60 min Heat at 95 C for 5 min to degrade the RNA and to inactivate the reverse transcriptase Add 80 ul of ddH2O to each 20 ul of cDNA synthesis reaction Mix well Hold the finished First Strand cDNA Synthesis Reaction on ice until the next step or store overnight at 20 C Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 13 C Performing Real Time PCR NOTE The use of SuperArray s RT Real Time SYBR Green PCR Master Mixes is absolutely critical for obtaining accurate results from the PCR Array Be sure to use the correct master mix for your instrument before continuing with this protocol See Pages 7 and 6 NOTE The use of the correct PCR Array plate format is also critical to the success of this experiment Be sure that you have the correct PCR Array format for your instrument before continuing with this protocol See Page 7 NOTE Accurate pipetting is very critical for the success of this protocol Be sure that all of your micro pipettors ar
28. ng 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 18S 28S Et MW RNA 28S n 8S MU BUD in o in Figure 3 Good Ribosomal RNA Band Integrity Is Important for Best Results from the PCR Array 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 above Technical Support 0800 2466651 49 40 853260 23 27 37 ts biomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 11 3 Genomic DNA Contamination Minimizing or eliminating genomic DNA contamination is essential for obtaining
29. ray by the average housekeeping gene value determined above for the same PCR Array 4 Determine the fold changes in relative gene expression Calculate the ratio between the background corrected housekeeping gene normalized numbers for each gene across two different PCR Arrays from two different samples 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 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 20 VI Troubleshooting and Frequently Asked Questions 1 The No Reverse Transcription control well H11 NRT yields a real time C value less than 35 cycles Contamination of the RNA with genomic DNA Be sure to include any DNase treatment steps in the recommended RNA isolation procedure or treat RNA separately with RNase free DNase followed by re purification using a spin column based method e g the Qiagen RNeasy Mini Kit 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 cont
30. rol 2 The No Template Control well H12 NTC yields a real time C value less than 35 cycles DNA contamination of other reagents tips and tubes See the Note on Preparing a Workspace Free of DNA Contamination at the beginning of the protocol in this User Manual 3 Will pipetting error affect the PCR Array results The passive reference dyes in the PCR 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 The use of standard multi channel pipettors will not affect the PCR Array results when passive reference dyes are used in PCR master mixes 4 How can lI 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 into your thermal cycler 5 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 duplicates or triplicates is highly recommended The Data Analysis Template available from our website for the PCR Array
31. s 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 1096 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 1096 bleach 4 Do not peel the protective film from the PCR Array plate until immediately ready to use Do not leave lab ware tubes and tip boxes exposed to the air for long periods of time Technical Support 0800 2466651 49 40 853260 23 27 37 ts Qbiomol de www SuperArray com RT Profiler PCR Array User Manual Version 1 5 4 21 2006 e 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 efficiency of if not block completely the enzyme act
32. ties 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 ReactionReady First Strand cDNA Synthesis 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 PCR 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 is a 96 well plate that includes primer sets for a thoroughly researched set of 84 relevant pathway or disease focused genes plus five housekeeping genes and two negative controls See Figure 2 for the layout of a typical PCR Array These primer sets and our master mixes have both been optimized hand in hand for SYBR Green real time 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
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