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RT2 Profiler PCR Array System

1. Cycles Duration Temperature 1 10 minutes 95C 45 15 seconds 95C 1 minute 60C Attention Roche LightCycler 480 Users Adjust the ramp rate to 1C sec Please refer to the Instrument Setup Guide at http sabiosciences com pcrarrayprotocolfiles php for more information on other REQUIRED changes to settings for Melt Curve Acquisition Use a three step cycling program for the following instrumentation Bio Rad Opticon Opticon 2 Chromo 4 MJ Research Takara TP 800 All other instruments Cycles Duration Temperature 1 10 minutes 95C 15 seconds 950 30 to 40 a seconds 396 30 seconds 726 1 The 10 minute step at 95 is required to activate the HotStart DNA polymerase 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 for your instrument NOTE For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www SABiosciences com pcrarrayprotocolfiles php Technical Support 888 503 3187 US 25 301 682 9200 RT Profiler PCR Arrays e Calculate the threshold cycle C for each well using the instrument s software NOTE For Roche LightCycler 480 Users there are two options available to analyze your d
2. 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 H2O 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 1 2 3 4 5 6 7 8 9 10 11 12 Samples Figure 5 Layout of the Housekeeping Genes PCR Arrays IFO DMoovODSs C 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 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 Technical Support 888 503 3187 US 301 682 9200 39 RT Profiler PCR Arrays 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 c
3. 28S U lt I8S NNU AUD V Ur o n A 1 U 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 Technical Support support SABiosciences com www SABiosciences com 16 Version 5 01 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 For reliable data from RT Profiler PCR Arrays amp RT qPCR Primer Assays a RNA Integrity Number RIN of 7 or higher is recommended NOTE Consistent RIN values across multiple samples within each experiment are desirable for reliable quality data comparisons NOTE Samples with degraded RNA such as from FFPE blocks slides with RINs less than 7 may be of sufficient quality but the final PCR Array Primer Assay or gene expression data must be checked for quality by the rese
4. by implication or by estoppel to use any instrument or system under any claim of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 other than for the amount of product contained herein Patent Pending TRADEMARKS RT Profiler PCR Array RT Real Time RT Nano PreAMP RT FFPE PreAMP XpressRef 384EZLoad SABiosciences and the SA logo are trademarks of SABiosciences Corporation ABI ROX StepOnePlus and ViiA 7 are registered trademarks of Applera Corporation Opticon 2 Chromo4 iQ5 iCycler CFX96 CFX384 and MyiQ are registered trademarks of Bio Rad Laboratories Inc LabChip is a registered trademark of Caliper Life Sciences LightCycler is a registered trademark of Roche Applied Sciences SmartCycler is a registered trademark of Cepheid SYBR is a registered trademark of Molecular Probes TRIzol is a registered trademark of Invitrogen Mastercycler is a registered trademark of Eppendorf Mx3000P Mx3005P and Mx4000 are registered trademarks of Stratagene RNeasy is a registered trademark of Qiagen N V Technical Support 888 503 3187 US 301 682 9200 41 RT Profiler PCR Arrays NOTES Technical Support support SABiosciences com www SABiosciences com 42 Version 5 01 NOTES Technical Support 888 503 3187 US 301 682 9200 43 RT Profiler PCR Array User Manual Part 1022A Version 5 01 9 16 2010 J SABiosciences A QIAGEN Company 1062756 7 1 2010 888 503 3187 301 682 9200 www SABiosciences com support S
5. support SABiosciences com www SABiosciences com 28 Version 5 01 3 Loading the Rotor Gene Q Rotor Disc 100 PCR Array NOTE Change pipet tips following each addition to avoid any cross contamination between the wells or reactions a CAREFULLY remove the PCR Array from its sealed bag and slide the array into the Rotor Disc 100 loading block using the tab at position A1 and the tube guide holes b Manually dispense 20 ul of the Experimental Cocktail into wells 1 through 100 of the PCR Array although wells 97 100 do not contain assays their loading is essential for optimized balancing of the Rotor Gene Q 100 PCR array Alternatively manual additional of the Experimental Cocktail can be avoided by using the QIAgility a compact benchtop instrument that provides rapid high precision PCR setup please contact a technical applications scientist at 1 888 503 3187or support SABiosciences com for further information c Proceed to the next section STEP 4 on Performing Real Time PCR Detection using the Rotor Gene Q 4 Performing Real Time PCR Detection using the Rotor Gene Q NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument f CAREFULLY seal the Rotor Gene Q 100 PCR Array with the Rotor Disc heat sealing film using the Rotor Disc heat sealer Allow one minute to pass and remove excess sealing film g Insert Rotor Gene Q 100 PCR Array into Rotor
6. Briefly 10 15 seconds spin down all reagents 2 Prepare the Genomic DNA Elimination Mixture a For each RNA sample combine the following in a sterile PCR tube Total RNA 25 0ngto5 0 ug GE 5X gDNA Elimination Buffer 2 0 uw H20 to a final volume of 10 0 uw The RT First Stand Kit C 03 is not compatible with the chemicals in Ambion s DNA free kits If your RNA sample has been treated with Ambion s DNA free reagents please call SABiosciences Technical Support at 1 888 503 3187 b Mix the contents gently with a pipettor followed by brief centrifugation c Incubate at 42 for 5 min d Chill on ice immediately for at least one minute 3 Prepare the RT Cocktail RT Cocktail 1 reaction 2 reactions 4 reactions BC3 5X RT Buffer 3 4 ul 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 H20 3 ul 6 ul 12 ul Technical Support 888 503 3187 US 301 682 9200 19 RT Profiler PCR Arrays 4 d e 5 First Strand cDNA Synthesis Reaction a Add 10 ul of RT Cocktail to each 10 ul Genomic DNA Elimination Mixture b C Mix well but gently with a pipettor Incubate at 42 for exactly 15 min and then immediately stop the reaction by heating at 95 for 5 minutes Add 91 ul of H2O 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
7. DNase treatment step RNase Free DNase Set 79254 Technical Support 888 503 3187 US 301 682 9200 15 RT Profiler PCR Arrays g 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 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 A260 A230 ratio should be greater than 1 7 ii A260 A280 ratio should be 1 8 to 2 0 iii Concentration by Azgo should be greater than 40 ug ml 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 1
8. E Fola Change Raton rea Tamer O OQOOOOHOOODOOOOO D OQOOHODOOOOOGHOO POODOOHOOOODOQOOOQOO Figure 1 Overview of the PCR Array Procedure Figure 2 Layout of the Cataloged Pathway Focused PCR Arrays Technical Support 888 503 3187 US 301 682 9200 5 RT Profiler PCR Arrays a y e co amp PAHS 01 1R 12 Z f few oe RT Pro fi a Human Intlammaro CR Array rA Receptors Ykines g Tet tag StOrO at apebiration Date 988502 31077 307 40 pes For research Fax 888 the the Rabe disc from the RAG DS T Geane N rani time haimaa a AG EE Figure 2C Layout of the Cataloged Pathway Focused Rotor Gene Q Rotor Disc 100 PCR Array Figure 2A Layout of the 96 Gene 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 The product information included with each cataloged PCR Array contains a list of the pathway focused and housekeeping genes on the array Housekeeping Assay Panel Wells H1 through H5 contain a housekeeping gene panel to normalize PCR Array data Genomic DNA Controls GDC Well H6 contains the Genomic DNA Control GDC Reverse Transcription Controls RTC Wells H7 through H9 contain replicate Reverse Transcription Controls RTC Positive PCR Controls PPC Wells H10 through H12 contain replicate Positive PCR Control
9. Minutes with the New GeneWeb Tool Go to http qeneweb sabiosciences com Technical Support 888 503 3187 US 301 682 9200 33 RT Profiler PCR Arrays 2 For 100 well R Format PCR Array on Rotor Disc 100 for Rotor Gene Q PCR Array Data Analysis Web Portal Excel amp Web based Utilities 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 33 or as N A not detected to 33 At this point any C value equal to 33 is considered a negative call 2 Examine the threshold cycle values of the control wells d Genomic DNA Control GDC Calculate C P If the value is greater than 33 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 33 then genomic DNA contamination is evident See the Troubleshooting and FAQ section e Reverse Transcription Control RTC Any impurities in your RNA sample that affect the reverse transcription
10. Support 888 503 3187 US 301 682 9200 23 RT Profiler PCR Arrays d Loading Custom Format PCR Arrays i CAREFULLY remove the PCR Array from its sealed bag ii Optional Dispense Experimental Cocktail to RT PCR Array Loading Reservoir PA 027 338162 to assist in loading iii Add each Sample Experimental Cocktail to each well of the PCR Array i 25 ul per well for 96 well Custom PCR Arrays ii 10 ul per well for 384 well Custom PCR Arrays iv Proceed to the next section STEP 4 on Performing Real Time PCR Detection 4 Performing Real Time PCR Detection ATTENTION Users of Bio Rad and Eppendorf Real Time Instruments Prior to initiating the run please make sure your instrument has been calibrated to use clear flat optical caps with PCR Array plates NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument a CAREFULLY but tightly 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 b Centrifuge the plate for 1 full minute at room temperature at 1000 g to remove bubbles Visually inspect the plate from underneath of the plate to ensure no bubbles are present in each well NOTE Bubbles remaining in the bottom of the wells of a PCR Array will interfere with results c Place the plate on ice while setting up the PCR cycling program below d Place one plat
11. 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 G60 eae 9 c Gol C HKG z gate 2 t 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 Atilexpt i gatean 2 Where AAC is equal to AC expt AC control The complete calculation is as follows g7ACGON expt 2 Mcontrol lt p MEKE control lt 2 central 7 ACAAKG control Technical Support support SABiosciences com www SABiosciences com 36 Version 5 01 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 Qiagen s RNeasy Mini Kit Catalog 74104 You must also then use SABiosciences s RT First Strand Kit C 03 330401 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 othe
12. of the RT First Strand Kit s built in external RNA control also affect the reverse transcription of your messages of interest Calculate AC AVG CPT AVG CPP If this value is less than 5 then no inhibition is apparent 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 support SABiosciences com www SABiosciences com 34 Version 5 01 f 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 value should be 14 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 inhibitors in each sample and that all of the RNA samples require further purification iii An average value of CP that is consistently greater than 16 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 AC Z Cee aCe HKG NOTE Choosing the right normalization factor The expression
13. on Page 8 Human RT RNA QC PCR Array Cat No PAHS 999 330291 Mouse RT RNA QC PCR Array Cat No PAMM 999 330291 Rat RT RNA QC PCR Array Cat No PARN 999 330291 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 338112 Mouse XpressRef Universal Total RNA Cat No GA 005 338114 Rat XpressRef Universal Total RNA Cat No GA 006 338116 FFPE Paraffin Block Applications a RNA Extraction Kit PA 023 330471 b FFPE PreAMP cDNA Synthesis Kit C 07 330461 c FFPE PreAMP Primer Mixes PFX 330151 RT PCR Array Loading Reservoir PA 027 338162 Technical Support support SABiosciences com www SABiosciences com 12 Version 5 01 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 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 componen
14. real time assays but will contain master mix to account for weight balance RT Profiler PCR Array Controls Definitions 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 efficiency 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 amp 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 888 503 3187 US 301 682 9200 7 RT Profiler PCR Arrays Il Materials Provided The PCR Arrays are available in 8 different plate formats each tailored to a specific subset of real time PCR instruments amp associated blocks Formats A C D and F are 96 well plates Formats E amp G are 384 well plates Format R are 100 well discs and Format H is 96 x 96 Chip compatible Format For Real Time Instruments Plate ABI standard blocks 5700 7000 7300 7500 7700 7900HT ViiA 7 96 well block A Bio R
15. resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with the SABiosciences PCR Array Data Analysis Template Excel or Web based Utilities ii Excel based PCR Array Data Analysis Templates available for 96 well 384 well 4x96 384HT and Custom PCR Array formats available at 1 http sabiosciences com pcrarraydataanalysis php iii Web based PCR Array Data Analysis Software at 1 http sabiosciences com pcrarraydataanalysis php Technical Support support SABiosciences com www SABiosciences com 26 Version 5 01 5 Recommended Quality Control Dissociation Melting Curve For instrument specific melt curve analysis settings please refer to the corresponding Instrument Setup Guide for your instrument at http sabiosciences com pcrarrayprotocolfiles php NOTE If you decide not to obtain the dissociation curve immediately save the plates wrapped in aluminum foil at 20 as is in case yo u 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 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 Re
16. ul of the cDNA synthesis reaction at 20 in case y ou need to perform one later for troubleshooting purposes CUSTOM PCR ARRAY NOTE Prepare Experimental Cocktail for each sample by calculating enough of each component based on the PCR Array layout selected Please prepare an excess of 10 for pipetting error Fluidigm BioMark Format H Plates Please refer to the RT cDNA Synthesis Kit BioMark Cat C 10 330431 for instructions on how to set up your plates 3 Loading the PCR Arrays Please select your PCR Array Format for loading instructions NOTE Change pipet tips following each addition to avoid any cross contamination between the wells or reactions NOTE f using robotic instrumentation for loading PCR Arrays please contact the SABiosciences Technical Support Team for PCR Array plate specifications a Loading the 96 Well PCR Array Formats A C D or F i CAREFULLY remove the PCR Array from its sealed bag ii Optional Dispense Experimental Cocktail to RT PCR Array Loading Reservoir PA 027 338162 to assist in loading iii 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 iv Proceed to the next section STEP 3 on Performing Real Time PCR Detection b Loading the 384 Well PCR Array Formats E or G NOTE Each 384 well plate characterizes four samples in separate sets of
17. use DEPC treated H20 Use high quality nuclease free H O If you are not sure whether your RNase DNase free water has been DEPC treated please check with the supplier NOTE f precipitates are present in the Master Mix tubes please contact a technical applications scientist at 1 888 503 31870r support SABiosciences com for further instructions NOTE Carefully pipette reagents from reagent tubes starting with pipette tip at top of tube and working down slowly 1 Briefly 10 15 seconds spin down all reagents 2 Experimental Cocktail Preparation for Custom PCR Arrays amp Plate H BioMark PCR Arrays see NOTES below Mix the following components in a 5 ml tube or a multi channel reservoir Plate Format 96 well 384 well 4x96 384HT Plate Format Designation A C D amp F E amp G E amp G 2X SABiosciences RT qPCR Master Mix 1350 wl 550 ul 2000 ul pe o A Strand cDNA Synthesis 102 ul 102 ul 102 ul H20 1248 ul 448 ul 1898 ul Total Volume 2700 ul 1100 ul 4000 ul NOTE This recipe provides an excess volume of ONLY 290 ul for the 96 well format to allow for multiple pipetting Very carefully add the cocktail to the PCR Array precisely as described below to ensure that each well receives the required volume Technical Support 888 503 3187 US 301 682 9200 21 RT Profiler PCR Arrays NOTE f you did not perform RNA quality control with a RT RNA QC PCR Array save the remainder of 9
18. 0 well RT Profiler PCR Arrays PA 042 12 330622 For 12 100 well RT Profiler PCR Arrays PA 042 24 330623 For 24 100 well RT Profiler PCR Arrays 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 two 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 4 Custom PCR Arrays 330131 Custom PCR Arrays are available in a number of formats allowing the customer to choose the number of genes to study and the number of sample replicates that may be needed Please select a master mix package size that provides sufficient volume for your samples E 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 or the Roche LightCycler 2 0 2 Calibrated Multi Channel Pipettor 3 RNase DNase free pipette tips and tubes Technical Support 888 503 3187 US 301 682 9200 11 RT Profiler PCR Arrays 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
19. 03 3187 US 301 682 9200 37 RT Profiler PCR Arrays 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 into 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 cha
20. 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 96 gene PCR Arrays are available in a 96 well plate format a 100 well Rotor Disc format and a 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 proc
21. 407 800 5 322 770 5 310 652 5 994 056 6 171 785 and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims in US Patent No 6 814 934 and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA This product is provided under an agreement between Molecular Probes Inc and SABiosciences and the manufacture use sale or import of this product is subject to one or more of U S Patent Nos 5 436 134 5 658 751 and corresponding international equivalents owned by Invitrogen Corp The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer where such research does not include testing analysi
22. 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 i CAREFULLY remove the PCR Array from its sealed bag ii Optional Dispense Experimental Cocktail to RT PCR Array Loading Reservoir PA 027 338162 to assist in loading iii 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 338125 and the figure below as a guide Technical Support support SABiosciences com www SABiosciences com 22 Version 5 01 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 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 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 Place Cover 4 red on the plate Add 10 uL of Sample 4 cocktail to the open wells Even number wel
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24. Disc 100 rotor Subsequently attach the Rotor Disc 100 locking ring h Insert loaded rotor with attached locking ring into Rotor Gene Q real time PCR machine NOTE PCR Arrays containing experimental cocktail may be store at 20 wrapped in aluminum foil for up to one week until ready to run i Enter and run the appropriate program below Cycles Duration Temperature 1 10 minutes 95C QIAGEN Rotor Gene Q 100 m 170 seconde 95C 30 seconds 60C 1 The 10 minute step at 95 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 NOTE For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www SABiosciences com pcrarrayprotocolfiles php Technical Support 888 503 3187 US 301 682 9200 29 RT Profiler PCR Arrays f Calculate the threshold cycle C for each well using the instrument s software NOTE For more details on data analysis parameters specific for the Rotor Gene Q please consult the Rotor Gene Q Manual iv To define the Baseline a Select Dynamic Tube default analysis setting to ensure the average background of each well is determined just before amplification commences b Optional Select Ignore First Fluorescent signal from the initial cycles may not be representative of the remainder of the run Thus better result
25. PA 384 for each PCR Array provided in the package NOTE Each 384EZLoad Cover is for a Single Use ONLY Technical Support support SABiosciences com www SABiosciences com Version 5 01 Shipping Conditions e PCR Arrays in formats A C D E F G and R are shipped at Room Temperature RT on Dry Ice DI or Blue Ice BI depending on destination amp accompanying products e PCR Arrays in format H are shipped on dry ice or blue ice packs Storage Conditions Keep plates and system components at 20 C for long term storage When stored properly at 20 their quality is gua ranteed for 6 months Technical Support 888 503 3187 US 301 682 9200 9 RT Profiler PCR Arrays B Additional Materials Required A RNA Isolation See Page 15 for specific recommendations High quality nuclease free H2O Do NOT USE DEPC H20O RT First Strand Kit Cat No C 03 330401 MANDATORY for a Complete and Successful Experiment For Reverse Transcription Control Detection RTC Wells H7 through H9 SABiosciences RT qPCR Master Mix MANDATORY for a Complete and Successful Experiment Be sure to pick the correct one for the instrumentation in your laboratory 1 96 Well amp 384 Well 96 x 4 Format Block PCR Arrays RT SYBR Green ROX qPCR Master Mix Specifically designed for e ABI 5700 7000 7300 7500 Standard amp FAST 7700 7900HT 96 well block Standard amp FAST and 384 well block StepOnePlus e Eppendorf M
26. Q and Custom PCR Array formats available at 1 http sabiosciences com pcrarraydataanalysis php ii Web based PCR Array Data Analysis Software at 1 http sabiosciences com pcrarraydataanalysis php Technical Support support SABiosciences com www SABiosciences com 30 Version 5 01 5 Recommended Quality Control Dissociation Melting Curve For Rotor Gene Q melt curve analysis settings please refer to the corresponding Instrument Setup Guide for your instrument at http sabiosciences com pcrarrayprotocolfiles php NOTE Be sure to visually inspect the Rotor Gene Q Ring 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 rings Removing 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 General Protocol 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 Melt curve analysis can be added during creation of the Rotor Gene Q qPCR program No more than one peak should appear in each reaction Technical Support 888 503 3187
27. SJ SABiosciences A QIAGEN Company User Manual Handbook 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 41 Part 1022A Version 5 01 9 16 2010 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 PARN PADM and PAQQ QIAGEN Catalog 330231 330111 330131 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 A QIAGEN Company 6951 Executive Way Suite 100 Frederick MD 21703 USA CONTENTS l Background and Introduction Il Materials Provided Ill Additional Materials Required IV Comp
28. US 301 682 9200 31 RT Profiler PCR Arrays D Data Analysis AAC Method 1 For 96 well amp 384 well Formats PCR Array Data Analysis Web Portal Excel amp Web based Utilities 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 threshold cycle values of the control wells a Genomic DNA Control GDC Calculate C E 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 o
29. ad iCycler iQ5 MyiQ MyiQ2 Chromo4 MJ Research Eppendort MasterCycler ep RealPlex 2 2s 4 4s Bonne Stratagene Mx3005p Mx3000p Takara TP 800 c ABI 7500 FAST block 7900HT FAST block StepOnePlus 96 well ViiA 7 FAST block D Bio Rad CFX96 Opticon and Opticon 2 MJ Research 96 well Stratagene Mx4000 ABI 7900HT 384 well block ViiA 7 384 well block i E Bio Rad CFX384 PERRO F Roche LightCycler 480 96 well block 96 well G Roche LightCycler 480 384 well block 384 well H Fluidigm BioMark 96x96 Chip 100 well R QIAGEN Rotor Gene Q Rotor Disc 100 NOTE The format of the PCR Array is indicated by the last letter of the catalog number Be sure that you have the correct PCR Array format for your instrument before starting the experiment The 96 well Plate and 100 well Disc PCR Arrays Formats A C D F and R are shipped in sets of 2 12 or 24 while the 384 well PCR Arrays Formats E and G are shipped in sets of 4 The PCR Array 384HT is shipped in sets of 2 12 or 24 The PCR Array Plate Format H is shipped in a Format A plate in solution on ice Each PCR Array shipment includes the arrays and either 12 optical thin wall 8 cap strips Formats A amp D 1 optical adhesive film Formats C E F and G or 1 Rotor Disc heat sealing film Format R per array Each 96x4 Format 384 Well PCR Array Formats E amp G also includes one set of 4 384EZLoad Covers Catalog
30. arch investigator 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 the RT RNA QC PCR Array 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 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 i Amount Considerations The PCR Array System yields results with as little as 25 ng or as much as 5 pg total RNA per array The new RT Nano PreAMP and RT FFPE PreAMP technologies allow fo
31. astercycler ep realplex 2 2S 4 4S e Stratagene Mx3000p Mx3005p Mx4000 e TaKaRa TP 800 Catalog Number Size PA 012 330520 For 2 96 well RT Profiler PCR Arrays PA 012 12 330522 For 12 96 well RT Profiler PCR Arrays PA 012 24 330523 For 24 96 well RT Profiler PCR Arrays PA 012 8 330521 For 4 384 well RT Profiler PCR Arrays RT SYBR Green Fluorescein qPCR Master Mix Specifically designed for e Bio Rad iCycler iQ5 MyiQ MyiQ2 Catalog Number Size PA 011 330510 For 2 96 well RT Profiler PCR Arrays PA 011 12 330512 For 12 96 well RT Profiler PCR Arrays PA 011 24 330513 For 24 96 well RT Profiler PCR Arrays PA 011 8 330511 For 4 384 well RT Profiler PCR Arrays RT SYBR Green qPCR Master Mix Specifically designed for instrumentation that does not require a reference dye e Bio Rad CFX96 CFX384 Chromo4 Opticon 2 e Roche LightCycler 480 96 well amp 384 well e All Others Catalog Number Size PA 010 330500 For 2 96 well RT Profiler PCR Arrays PA 010 12 330502 For 12 96 well RT Profiler PCR Arrays PA 010 24 330503 For 24 96 well RT Profiler PCR Arrays PA 010 8 330501 For 4 384 well RT Profiler PCR Arrays 2 100 Well Ring PCR Array for QIAGEN Rotor Gene Q 100 R Plate Format Rotor Disc 100 RT FAST SYBR Green ROX qPCR Master Mix Specifically designed for e QIAGEN Rotor Gene Q Technical Support support SABiosciences com www SABiosciences com 10 Version 5 01 Catalog Number Size PA 042 330620 For 2 10
32. ata 1 Use the second derivate max setting amp there is no need to set a threshold OR 2 Use Fit Points and follow instruction ii below To define the Baseline choose the Automated Baseline option if your instrument has the Adaptive Baseline Function check with instrument manual or manufacturer if unsure If it does not have the adaptive baseline function you will need to set the baseline manually Use the Linear View of the amplification plots to determine the earliest visible amplification Set the instrument to use the readings from cycle number two 2 through two 2 cycles before the earliest visible amplification but no more than cycle 15 The earliest amplifications usually will be visible between cycles 14 and 18 Manually define the Threshold Value by using 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 IMPORTANT Ensure that the thresholds are the same across all PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays If the RNA sample quality has been adequately controlled the cycling program has been executed properly and the thresholds have been defined correctly then the value of C should be 20 2 across all of your arrays or samples If not see the Troubleshooting and FAQ section Export the
33. e 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 store at 20 wrapped in aluminum foil for up to one week until ready to run e Enter and run the appropriate program for your real time instrument below If prompted by your instrument software select Absolute Quantitation to begin Technical Support support SABiosciences com www SABiosciences com 24 Use a two step cycling program for the following instrumentation Version 5 01 ABI 5700 7000 7300 7500 7700 7900HT StepOnePlus ViiA 7 Bio Rad iCycler iQ5 MyiQ MyiQ2 CFX96 CFX384 Eppendorf Mastercycler ep realplex 2 28 4 4S Stratagene Mx3000p Mx3005p Mx4000p Cycles Duration Temperature 1 10 minutes 95C 15 seconds 95C ii 1 minute 60 Attention Bio Rad CFX96 amp CFX384 Users e Adjust the ramp rate to 1 C sec Attention Eppendorf Mastercycler ep realplex 2 2S 4 and 4S Users e For the Silver Thermoblock Adjust the ramp rate to 26 e For the Aluminum Thermoblock Adjust the ramp rate to 35 e Please refer to the Instrument Setup Guide at http sabiosciences com pcrarrayprotocolfiles php for detailed setup instructions Use an extended two step cycling program for the following instrumentation Roche LightCycler 480
34. edure provides high sensitivity and wide dynamic range Designed for Routine Use Bring expression profiling to 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 Version 5 01 How It Works 2A Standard PCR Array Layouts Isolate RNA from your Experimental Samples 96 well PCR Array Start with as little e ng of rt 1 ug is recommended treat wit ase a 000000000000 AAR DO Foo Add cDNA to RT qPCR Master Mix RT qPCR Master Mixes contain SYBR Green and reference dye 30 minutes minutes cDNA at cDNA sate y PCR Mix SS PCRMix2 Sex i Sample Loading Aliquot the Mixture Across Your PCR Arrays Order 384 well PCR Array 4x96 Each PCR Array profiles the expression of 84 pathway specific genes plus controls PCR Array 1 PCR Array 2 R 2 n Ss Mi Perform Thermal Cycling Collect real time amplification data using your instrument s software 2 Profile 1 Profile 2 2 10 900 10 000 co e 10 N y fa gt 0 c CAN i 1 eka 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 n 2 ele fof Change ee ae Z 5
35. eparing a Workspace Free of DNA Contamination For accurate and reproducible PCR Array results it is very important to avoid contamination of the assay with foreign DNA Any DNA contamination will artificially inflate the SYBR Green signal yielding skewed gene expression profiles and false positive signals The most common sources of DNA contamination are the products of previous experiments spread into the air of your working environment Please follow the recommendations below on how to set up and maintain a working environment free of DNA contamination 1 Wear gloves throughout the procedure Use only fresh PCR grade reagents H20 and lab ware tips and tubes 2 Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate your PCR workspace and lab ware pipettor barrels tube racks etc before each new use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 10 bleach to chemically inactivate and degrade any DNA 3 Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any lab ware tips or tubes containing PCR products or other DNA treat with 10 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 previ
36. f interest Calculate AC AVG CPT AVG C PP If this value is less than 5 then no inhibition is apparent 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 NOTE For RT Nano PreAMP and RT FFPE PreAMP Users Please refer to corresponding user manuals for correct settings and GDC PPC and RTC values Technical Support support SABiosciences com www SABiosciences com 32 Version 5 01 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 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 inhibitors in each sample and that all of the RNA samples require further purification iii 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 AC Ci GOI Gi AVG HKG NOTE Choosing the right n
37. hosen 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 support SABiosciences com www SABiosciences com 40 Version 5 01 RT Profiler PCR Arrays NOTICE TO PURCHASERS This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use Use of kit components for reproduction of any primer pair mix to modify kit components for resale or to use RT Profiler PCR Arrays to manufacture commercial products without written approval of SABiosciences Corporation is expressly prohibited PRODUCT WARRANTY This warranty limits our liability to the replacement of 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 LIMITED LICENSE STATEMENTS Use of this product is covered by one of more of the following US patents and corresponding patent claims outside the US 5 079 352 5 789 224 5 618 711 6 127 155 5 677 152 claims 1 to 23 only 5 773 258 claims 1 and 6 only 5
38. ion technique examine the quality of your RNA before this step using SABiosciences s species and instrument specific RT RNA QC PCR Arrays See Page 15 NOTE Do not use DEPC treated H20 Use high quality nuclease free H O If you are not sure whether your RNase DNase free water has been DEPC treated please check with the supplier NOTE f precipitates are present in the Master Mix tubes please contact a technical applications scientist at 1 888 503 31870r support SABiosciences com for further instructions NOTE Carefully pipette reagents from reagent tubes starting with pipette tip at top of tube and working down slowly 1 Briefly 10 15 seconds spin down all reagents 2 Experimental Cocktail Preparation Mix the following components in a 5 ml tube Array Format 100 well Array Format Designation R 2X SABiosciences RT Fast SYBR Green Rox PCR Master Mix 1150 ul Diluted First Strand cDNA Synthesis Reaction 102 ul H20 1048 ul Total Volume 2300 ul NOTE This recipe provides an excess volume of ONLY 300 ul for the 100 well R format Very carefully add the cocktail to the PCR Array precisely as described below to ensure 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 remainder of 9 ul of the cDNA synthesis reaction at 20 in case y ou need to perform one later for troubleshooting purposes Technical Support
39. irst Strand Kit C 03 330401 NOTE The use of SABiosciences s RT First Strand Kit Cat No C 03 330401 is critical for detecting the Reverse Transcription Controls RTC Wells H7 H9 and for obtaining the best results from the PCR Array See Pages 11 and 15 for more information NOTE RNA samples must meet the standards of integrity and purity from protein organics and genomic DNA contamination described on the previous two pages NOTE Use the same amount of total RNA in this reaction for every sample First time users are recommended to start with 1 0 ug of total RNA for 96 well plate format 0 8 ug of total RNA for 100 well Rotor Disc format 400 ng of total RNA for 384 well 4 x 96 plate format and 1 0 ug of total RNA for a 384 well HT plate format PCR Array Lower amounts of total RNA than 100 ng will dramatically increase the false negative rate of the PCR Array method NOTE Scientists using the Fluidi igm BioMark Real Time PCR System Please refer to the specification sheet for the RT First Strand Kit BioMark Cat C 10 330431 for first strand cDNA synthesis protocol and for sample plate preparation instructions NOTE Do not use DEPC treated H20 Use high quality nuclease free H O If you are not sure whether your RNase DNase free water has been DEPC treated please check with the supplier NOTE Carefully pipette reagents from reagent tubes starting with pipette tip at top of tube and working down slowly 1
40. lementary Products V Protocol A RNA Preparation and Quality Control B RT First Strand Kit C Performing Real Time PCR a For 96 well and 384 well Formats b For 100 well Rotor Gene Q 100 D Data Analysis a For 96 well and 384 well Formats b For 100 well Rotor Gene Q 100 VI Troubleshooting and Frequently Asked Questions Appendix Modified Protocol for Housekeeping Gene PCR Arrays 10 12 13 15 19 21 28 32 34 37 39 RT Profiler PCR Arrays 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 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
41. 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 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 Technical Support 888 503 3187 US 301 682 9200 35 RT Profiler PCR Arrays 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
42. ls of rows B D F H J L N amp P Remove amp discard the cover Sample 2 4 2 3 5 6 7 8 10 14 22 13 44 25 26 47 28 19 20 24 22 23 2 o Bele tE a Ead kad ba ko LE baal LALI LSI alo gt VOl S SIrl R C TIO mM M O a oa sy 4123 4 5 617 8 9 10 47 22 23 14 15 16 47 18 49 20 24 22 23 24 Sample 4 4 12 3 4 5 61 71 81 9 70 47 42 43 14 15 16 17 18 19 20 24 22 23 24 Ble tE a ead kad a TIO MMO alo gt VOl S SIrl R C TIO MI M O a go se 41213 4 5 617 8 8 20 47 22 23 14 15 26 47 18 29 20 24 22 23 24 C 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 ii Proceed to the next section STEP 3 on Performing Real Time PCR Detection Loading the PCR Array 384HT i CAREFULLY remove the PCR Array from its sealed bag ii Optional Dispense Experimental Cocktail to RT PCR Array Loading Reservoir PA 027 338162 to assist in loading iii Add 10 ul of the Experimental Cocktail to each well of the PCR Array preferably from a reservoir with an 8 channel pipettor or a 12 channel pipettor but only using 8 tips iv Proceed to the next section STEP 3 on Performing Real Time PCR Detection Technical
43. moving 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 General Protocol 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 If your instrument does not have a defau It melting curve program run the following program instead 95 1 min 65 2 min OPTICS OFF 65 to 95T at2 min OPTICS ON Technical Support 888 503 3187 US 301 682 9200 27 RT Profiler PCR Arrays 2 100 well R Format PCR Array on Rotor Disc 100 for Rotor Gene Q NOTE The use of SABiosciences s RT FAST 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 9 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 isolat
44. nges in gene expression 4 Can I use cDNA synthesized from a first strand kit from another manufacturer If your cDNA template is not synthesized using an SABiosciences RT First Strand Kit cDNA from 500 ng to 5 pg total RNA may be used If the cDNA synthesis reaction volume was o 20 ul or more add H20 to the synthesized cDNA to increase the volume to 111 ul and proceed as directed in Section B Step 4e o less than 20 ul add H2O to the synthesized cDNA to increase the volume to 20 ul and proceed as directed in Section B Step 4d 5 Do I need to prepare my reactions or pre mix on ice You can prepare your reactions at room temperature Since the SABiosciences RT Master Mixes include a Hot Start DNA Polymerase that is active only upon heat activation you can be assured that no non specific amplification results will be produced 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 Technical Support support SABiosciences com www SABiosciences com 38 Version 5 01 Appendix Modified Protocol for Housekeeping Gene PCR Arrays A 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 B Perform Real Time PCR
45. olume of reagent at least ten times greater than the tissue volume ii After the ethanol precipitation step further clean up the RNA using the Qiagen RNeasy Mini Kit Catalog 74104 e You must perform the recommended on column DNase treatment step RNase Free DNase Set 79254 c Formalin fixed Paraffin embedded FFPE Samples i Please refer to protocol for SABiosciences RT FFPE RNA Extraction Kit Catalog PA 023 330471 d Small Samples Yielding Less than 100 ng Total RNA i Please refer to protocol for Molecular Devices PicoPure RNA Isolation Kit Catalog KIT0204 e Whole Blood Samples i 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 ii The white plood cell fraction is then used for RNA isolation with the Qiagen RNeasy Mini Kit Catalog 74104 e You must perform the recommended on column DNase treatment step iii Alternatively the PAXgene Blood RNA Kit Qiagen Catalog 762164 can also be used to prepare total RNA from whole blood samples f Total RNA Isolated Using a Phenol Based Method If you have already prepared total RNA from any biological source material using a phenol based method such as QlAzol TRIzol RNAzol etc you must clean up the RNA with the Qiagen RNeasy Mini Kit Catalog 74104 e You must perform the recommended on column
46. ormalization 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 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 Optional Go From Fold Change to Biology in
47. ously 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 support SABiosciences com www SABiosciences com 14 Version 5 01 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 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 the Qiagen RNeasy Mini Kit Catalog 74104 e You must perform the recommended on column DNase treatment step RNase Free DNase Set 79254 b Tissue Samples i First extract RNA from the tissue using the QIAzol TRIzol protocol Be sure to use a sufficient amount of QIAzol TRIzol reagent During homogenization add a v
48. r gene expression analysis from as little as 1 ng of total RNA or 100 ng RNA from FFPE samples Please refer to the corresponding user manuals for additional information The optimal amount of starting material depends on the relative abundance of the transcripts of interest Lower abundance transcripts require more RNA higher Technical Support 888 503 3187 US 301 682 9200 17 RT Profiler PCR Arrays 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 330401 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 1 0 wg of total RNA for 96 well plate format 0 8 ug of total RNA for 100 well Rotor Disc format 400 ng of total RNA for 384 well 4 x 96 plate format and 1 0 ug of total RNA for 384 well HT plate format PCR Array 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 18 Version 5 01 B RT F
49. r 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 soectrophotometry in RNase free Tris pH 8 0 buffer If necessary re purify your RNA samples with a spin column based clean up method such as Qiagen s RNeasy Mini Kit Catalog 74104 with the subsequent RNase Free DNase Set 79254 3 Improving Poor PCR Amplification Efficiency Different instruments have different levels of sensitivity If an average CPP value of 20 2 is difficult to obtain for your instrument the observed average C PP 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 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 888 5
50. s PPC The 384 well 4 x 96 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 Technical Support support SABiosciences com www SABiosciences com 6 Version 5 01 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 The product information included with each cataloged PCR Array contains a list of the pathway focused and housekeeping genes on the array Housekeeping Assay Panel Wells P12 through P15 contain a housekeeping gene panel to normalize PCR Array data Genomic DNA Controls GDC Wells P16 through P18 contain replicate Genomic DNA Controls GDC Reverse Transcription Controls RTC Wells P19 through P21 contain replicate Reverse Transcription Controls RTC Positive PCR Controls PPC Wells P22 through P24 contain replicate Positive PCR Controls PPC Figure 2C Layout of the 100 Well Rotor Gene Q Rotor Disc 100 PCR Array The 96 real time assays found in the 96 well PCR plate based arrays are located in wells 1 through 96 of the Rotor Gene Q ring plate A1 A12 gt Ring 1 12 plate B1 B12 gt Ring 13 24 etc To maintain data analysis compatibility wells 97 100 do not contain
51. s may be achieved if the initial cycles are ignored Up to 5 cycles can be ignored c Optional Select Noise Slope Correction Selection of this option can improve data whose baseline initial cycles is noticeably sloped Noise Slope Correction improves the data when raw data backgrounds are observed to slope upward or downward before the takeoff point C IMPORTANT Ensure that all selections remain consistent across all PCR Array runs in the same analysis v Manually define the Threshold Value by using 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 IMPORTANT Ensure that the thresholds are the same across all PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays If the RNA sample quality has been adequately controlled the cycling program has been executed properly and the thresholds have been defined correctly then the value of CPP should be 14 2 across all of your arrays or samples If not see the Troubleshooting and FAQ section vi Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with the SABiosciences PCR Array Data Analysis Template Excel or Web based Utilities i Excel based PCR Array Data Analysis Templates available for 96 well 384 well 4x96 384HT Rotor Gene
52. s or screening services for any third party in return for compensation on a per test basis The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product of its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 The purchase of this product includes a limited non transferable license under specific claims of U S Patent Nos 6 174 670 6 569 627 and 5 871 908 owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology Inc and Roche Diagnostics GmbH to use only the enclosed amount of product according to the specified protocols No right is conveyed expressly
53. store overnight at 20 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 Technical Support support SABiosciences com www SABiosciences com 20 Version 5 01 C Performing Real Time PCR 1 96 well amp 384 well Block Formats 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 9 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 See Page 15 NOTE Do not
54. ts 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 products 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 888 503 3187 US 301 682 9200 13 RT Profiler PCR Arrays NOTE Pr

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