manual, GeneChip 3' IVT Express User Manual
Contents
1. 27 Target Hybridization for HT Array Plates Processed on the GeneTitan HVS CUMING TOIL ce P 30 Prepare the Hybridization Cocktail Master Mix 30 Hybridization Setup 32 GeneChip 3 IVT Express Kit User Manual Appendix A Appendix B Appendix C Troubleshooting 60k eee ada donc eR KK KB KK x CR REOR 35 Positive Control Reaction i s 444 52 A4 kb a la AKNG S REPRE ERES 35 Control RNA Amplification Instructions 35 Expected RESULTS ER AE 35 Factors that Affect Both Positive Control and Experimental Samples 35 Troubleshooting Low Yield and Small Average aRNA Size 36 aRNA Purification Photos x cnc ges KK OS 37 Shaker Speeds iius aa her KK kK KK KK KK KK KK KK KK eut s 39 Overview The GeneChip 3 IVT Express Kit is the latest technology in RNA target preparation for microarray expression analysis This kit features m Low RNA input requirements from as little as 50ng of total RNA for a single round of amplification Streamlined workflow with the option to decrease target labeling time to a single day with appropriate inputs of total RNA Master mixes consumables included and a simple protocol for ease of use convenience and a high rate of success A complete kit that includes Poly A RNA controls and hybridization controls Magnetic bead aRNA purification for high recovery and ease of2. Legend INENG RNA 0 0 DNA SM T7 promoter Biotin 4 GeneChip 3 IVT Express Kit User Manual Kit Contents and Storage Conditions Table 1 1 GeneChip 3 IVT Express Kit Components and Storage Conditions Component Vol Qnty Vol Qnty Storage 10 Rxn 30 Rxn BOX 1 of 2 aRNA Binding Buffer Concentrate 600 uL 1 8 mL room temp RNA Binding Beads 120 uL 360 uL 2 8 C aRNA Wash Solution Concentrate Add 8 mL 10 mL 10 mL room temp 100 ethanol before use as shown on the label aRNA Elution Solution 5 mL 5 mL room temp Nuclease free Water 10 mL 10 mL room temp 5X Array Fragmentation Buffer 1 mL 1 mL room temp 8 Strip PCR Tubes amp Caps 0 2 mL 10 ea 20 ea room temp U Bottom Plate 1 ea 2 ea room temp Reservoir 1 ea 1 ea room temp BOX 2 of 2 First Strand Enzyme Mix 11 uL 33 uL 20 C First Strand Buffer Mix 44 uL 132 uL 20 C Second Strand Enzyme Mix 22 uL 66 uL 20 C Second Strand Buffer Mix 55 uL 165 uL 20 C IVT Enzyme Mix 66 uL 198 uL 20 C IVT Labeling Buffer 220 uL 660 uL 20 C IVT Biotin Label 44 uL 132 uL 20 C Control RNA 1 mg mL HeLa total RNA 10 uL 10 uL 20 C Nuclease free Water 1 75 mL 1 75 mL 20 C Poly A Control Stock 16 uL 16 uL 20 C Poly A Control Dilution Buffer 3 8 mL 3 8 mL 20 C 20X Hybridization Controls 450 uL 450 uL 20 C Control Oligo B2 150 uL 150 uL 20 C Do not freeze
3. B Vigorously shake the plate for 3 minutes setting 10 on the Lab Line Titer Plate Shaker Then check to make sure the RNA Binding Beads are fully dispersed If they are not continue shaking until the beads are dispersed C Move the plate to a magnetic stand and capture the RNA Binding Beads D Transfer the supernatant which contains the eluted aRNA to a nuclease free PCR tube 7 Store aRNA at lt 20 C or place on ice and proceed with quantitation and fragmentation Purified aRNA can be stored at 20 C for up to 1 year As with any RNA preparation the number of freeze thaw cycles should be minimized to maintain aRNA integrity Evaluation and Fragmentation of aRNA aRNA Quantitation and Expected Yield Assessing aRNA Yield by UV Absorbance The concentration of an aRNA solution can be determined by measuring its absorbance at 260 nm We recommend using NanoDrop Spectrophotometers for convenience No dilutions or cuvettes are needed just measure 2 uL of the aRNA sample directly Alternatively the aRNA concentration can be determined by diluting an aliquot of the preparation in TE 10 mM Tris HCl pH 8 1 mM EDTA and reading the absorbance in a traditional spectrophotometer at 260 nm Find the concentration in ng mL using the equation shown below 1 A 40 ug RNA mL Assessing aRNA Yield With RiboGreen A X dilution factor X 40 ug RNA mL If a fluorometer or a fluorescence microplate reader is available
4. Materials Required Reagents Table 1 2 Reagents Chapter 1 Overview Material Source P N GeneChip IVT Express Kits See Table 1 1 for detailed kit information Affymetrix 901228 10 Rxn 901229 30 Rxn containing Hybridization Module from Box 1 o Pre Hybridization Mix o 2X Hybridization Mix a DMSO o Nuclease free water Stain Module from Box 1 a Stain Cocktail 1 a Stain Cocktail 2 n Array Holding Buffer Wash Buffers A and B from Box 2 o Wash Buffer A P N 900721 a Wash Buffer B P N 900722 GeneChip Hybridization Wash and Stain Kit cartridge arrays Affymetrix 900720 30 Rxn HT array plates containing Box 1 of 2 o 1 3X Hybridization Solution A a 1 3X Hybridization Solution B a Stain Cocktail 1 amp 3 a Stain Cocktail 2 n Array Holding Buffer Box 2 of 2 a Wash Buffer A a Wash Buffer B GeneTitan Hybridization Wash and Stain Kit for 3 IVT Arrays Affymetrix 901530 10096 ethanol ACS reagent grade multiple Quant iT RiboGreen RNA Reagent Optional Invitrogen R11490 For hybridization washing and staining of the targets prepared using the GeneChip 3 IVT Express Kit onto GeneChip arrays users should purchase the GeneChip Hybridization Wash and Stain Kit For hybridization washing and staining of the targets prepared using the GeneChip 3 IVT Express Kit onto HT array plates users should purchase the GeneTitan Hybrid
5. m The positive control reaction should produce gt 50 ug of aRNA u The average size of the aRNA should be 800 nucleotides Factors that Affect Both Positive Control and Experimental Samples If the positive control reaction yield or amplification product size does not meet expectations consider the following possible causes and troubleshooting suggestions These suggestions also apply to problems with amplification of experimental RNA Incubation Temperature s Were Incorrect The incubation temperatures are critical for effective RNA amplification Use only properly calibrated thermal cyclers for the procedure Condensation formed in the tube during the reaction incubation s Condensation occurs when the cap of the reaction vessel is cooler e g room temperature than the bottom of the tube As little as 1 2 uL of condensate in an IVT reaction tube throws off the concentrations of the nucleotides and magnesium which are crucial for good yield If you see condensation check to make sure that the heated lid feature of the thermal cycler is working properly 36 GeneChip 3 IVT Express Kit User Manual Nuclease Contamination Using pipettes tubes or equipment that are contaminated with nucleases can cleave the RNA or DNA being generated at each step in the procedure This will reduce the size of the aRNA products and decrease aRNA yield Both RNases and DNases can be removed from surfaces using a RNase decontamination solutio
6. In Vitro Transcription to Synthesize Biotin Modified aRNA with IVT Labeling Master Mix generates multiple copies of biotin modified aRNA from the double stranded cDNA templates this is the amplification step aRNA Purification removes unincorporated NTPs salts enzymes and inorganic phosphate to improve the stability of the biotin modified aRNA Fragmentation of the labeled aRNA prepares the target for hybridization to GeneChip 3 expression arrays Use the included Control RNA to familiarize yourself with the GeneChip 3 IVT Express Kit RNA Amplification procedure Instructions for the positive control reaction are provided in Appendix A Troubleshooting on page 35 Chapter 1 Overview 3 Figure 1 1 Overview of the GeneChip 3 IVT Express Kit Labeling Assay Approximate Total RNA Sample Time 58 E8480 1018108010 BAAAAA G Poly A RNA Controls 1 Poly A RNA Conttrol x d a T7 Oligo dT Primer 3 TTTTT WI 5 2 First strand x esie cDNA Synthesis 5 TTITITI N PAAAAA 3 3 ll ll Il lI 00 0 0 I TTTTT m 5 3 Second strand cDNA Synthesis vost 1 5 hours 5 TTTTTTTTUTTT AAAAA azm 3 s 00000 L11111TTTTT Ell 5 4 IVT Labeli Biotinylated f Song Ribonucleotide 4 hours or Brann v x Analog 16 hours 3 LI II II L L TUUUUU 5 voor 3 I II111111 1UUUUU 5 AN E RAE 3 L ILILI _ILILLLILL 1UUUUU 5 5 aRNA Purification deis 6 Fragmentation 1 hour 7 Hybridization 16 hours
7. 2001 The actual amount of mRNA depends on the cell type and the physiological state of the sample When calculating the amount of amplification the starting mass of mRNA in a total RNA prep should always be considered within a range of 10 30 ng per ug of total RNA assuming good RNA quality aRNA Purification Photos UJ Figure B 1 Photos of aRNA Purification Step figure 1 of 2 Ambion Magnetic Stand 96 P N AM10027 Magnetic Stand aRNA Binding Step After addition of ethanol and mixing RNA Binding Beads Capture After 5 minutes on magnetic stand Ambion 96 well Magnetic Ring Stand P N AM10050 38 GeneChip 3 IVT Express Kit User Manual Ambion Magnetic Stand 96 P N AM10027 Bead Washing After second wash and 1 minute shake Removal of Ethanol Dry beads following 1 minute shake aRNA Elution Dispersed beads following 3 minute shake Elution Step Recovery of purified aRNA following bead capture Ambion 96 well Magnetic Ring Stand P N AM10050 Shaker Speeds Table C 1 Plate Shaking Speeds aRNA Binding Gentle 300 500 4 1 Bead Washing Moderate 700 900 7 4 Ethanol Removal Vigorous 1000 1200 10 7 aRNA Elution Vigorous 1000 1200 10 7 1200 1000 oo e o Approximate RPM a e e LI A e o 200 0 1 2 3 4 5 6 7 8 9 10 Speed Setting 40 GeneChip 3 IVT Express Kit User M
8. Components of the GeneChip IVT Express Kit box 1 are used for this step Fragmentation of aRNA target before hybridization onto GeneChip probe arrays has been shown to be critical in obtaining optimal assay sensitivity Affymetrix recommends that the aRNA used in the fragmentation procedure be sufficiently concentrated to maintain a small volume during the procedure This will minimize the amount of magnesium in the final hybridization cocktail Fragment an appropriate amount of aRNA for hybridization cocktail preparation and gel analysis aRNA amount depends on the format of the GeneChip probe array you are using 1 Assemble the aRNA fragmentation mixture Table 3 1 Sample Fragmentation Reaction by Array Format Component 49 64 Format 100 Format 169 400 HT Format aRNA 15 ug 1 to 32 uL 12 ug 1 to 25 6 uL 7 5 ug 1 to 16 uL 5x Array Fragmentation Buffer 8 uL 6 4 uL 4 uL Nuclease free Water Variable up to Variable up to Variable up to 40 uL final volume 32 uL final volume 20 pL final volume Total Volume 40 pL 32 pL 20 pL Please refer to specific probe array package insert for information on array format 2 Fragmentation Reaction A Incubate the fragmentation reaction at 94 C for 35 minutes B Place the reaction on ice immediately after the incubation 3 Optional Evaluate a sample of the reaction on a Bioanalyzer Analyze the size of the fragmentation reaction products by running a 300 ng s
9. Components of the GeneChip IVT Express Kit box 2 are used for this step 1 Assembly of First Strand Master Mix A Thaw first strand synthesis reagents and place on ice B On ice assemble First Strand Master Mix in a nuclease free tube in the order listed in Table 2 7 Include 5 overage to cover pipetting error Table 2 7 First Strand Master Mix for a single reaction Component Amount First Strand Buffer Mix 4uL First Strand Enzyme Mix 1 uL Total Volume 5 uL C Mix well by gently vortexing Centrifuge briefly 5 seconds to collect the mix at the bottom of the tube D Place the supplied PCR Tubes or Plate on ice and transfer 5 uL First Strand Master Mix to individual tubes or wells 2 Addition of Total RNA poly A Control Mixture A Add 5 uL of the Total RNA poly A Control Mixture Table 2 6 to each aliquot of First Strand Master Mix for a final volume of 10 uL B Mix thoroughly by gently vortexing Centrifuge briefly to collect the reaction at the bottom of the tube plate and place on ice 3 Incubation A Incubate for 2 hours at 42 C in a thermal cycler using the program for First Strand cDNA Synthesis Table 2 3 on page 12 B After the incubation centrifuge briefly 5 seconds to collect the first strand cDNA at the bottom of the tube plate Place the sample on ice and immediately proceed to Second Strand cDNA synthesis below 16 GeneChip 3 IVT Express Kit User Manual Second
10. Strand cDNA Synthesis Components of the GeneChip IVT Express Kit box 2 are used for this step 1 Assembly of Second Strand Master Mix A On ice prepare a Second Strand Master Mix in a nuclease free tube in the order listed in Table 2 8 Prepare master mix for all the samples in the experiment including 5 overage to cover pipetting error Table 2 8 Second Strand Master Mix for a single reaction Component Amount Nuclease free Water 13 uL Second Strand Buffer Mix 5 uL Second Strand Enzyme Mix 2 uL Total Volume 20 pL B Mix well by gently vortexing Centrifuge briefly 5 seconds to collect the mix at the bottom of the tube and place on ice C Transfer 20 uL Second Strand Master Mix to each 10 uL cDNA sample Mix thoroughly by gently vortexing or flicking the tube 3 4 times Centrifuge briefly to collect the reaction at the bottom of the tube plate and place on ice D Place the reaction in a 16 C thermal cycler block It is important to pre cool the thermal cycler block to 16 C because subjecting the reaction to temperatures 716 C will compromise aRNA yield 2 Incubation A Incubate for 1 hour at 16 C followed by 10 minutes at 65 C in a thermal cycler using the program for Second Strand cDNA Synthesis Table 2 3 on page 12 NOTE Cover reactions with the heated lid of the thermal cycler even if its temperature cannot be adjusted to match the block temperature B After the in
11. and extract the Pre Hybridization Mix from the array with a micropipettor Refill the array with the appropriate volume of the clarified hybridization cocktail avoiding any insoluble matter at the bottom of the tube see Table 4 2 Place probe array into the hybridization oven set to 45 C 10 To avoid stress to the motor load probe arrays in a balanced configuration around the axis Rotate at 60 rpm 11 Hybridize for 16 hours NOTE During the latter part of the 16 hour hybridization prepare reagents for the washing and staining steps required immediately after completion of hybridization For further instruction please refer to u Affymetrix GeneChip Fluidics Station 450 250 User Guide P N 08 0092 u GeneChip Expression Analysis Technical Manual with Specific Protocols for Using the GeneChip Hybridization Wash and Stain Kit P N 702232 u GeneChip Expression Wash Stain and Scan Manual for Cartridge Arrays P N 702731 30 GeneChip 3 IVT Express Kit User Manual Target Hybridization for HT Array Plates Processed on the GeneTitan Instrument This section provides instruction for setting up HT array plate hybridizations using the GeneTitan Hybridization Wash and Stain Kit for 3 IVT Arrays For ordering information please refer to Table 1 2 on page 5 Prepare the Hybridization Cocktail Master Mix Reagents and Materials Required u GeneTitan Hybridization Wash and Stain Kit for 3 IVT Arrays
12. not equilibrated to room temperature they may be prone to cracking which can lead to leaks 3 Heat the hybridization cocktail to 99 C for 5 minutes in a heat block 4 Meanwhile wet the array with an appropriate volume of Pre Hybridization Mix Table 4 2 by filling it through one of the septa Table 4 2 Probe Array Cartridge Volumes for Pre Hybridization Mix and Hybridization Cocktail Array Volume 49 Format Standard 200 uL 64 Format 200 uL 100 Format Midi 130 uL 169 Format Mini 80 uL 400 Format Micro 80 uL NOTE Each array has two septa see Figure 4 1 for location of the probe array septa In order to fill the array first vent the array chamber by inserting a clean unused pipette tip into one of the septa then insert the pipette tip of a micropipettor into the remaining septum to fill Chapter 4 Hybridization 29 Figure 4 1 GeneChip Probe Array Plastic cartridge Front Probe array on glass substrate 9 Incubate the probe array filled with Pre Hybridization Mix at 45 C for 10 minutes with rotation Transfer the hybridization cocktail that has been heated at 99 C in Step 3 to a 45 C heat block for 5 minutes Spin the hybridization cocktail at maximum speed in a microcentrifuge for 5 minutes to collect any insoluble material from the hybridization mixture Remove the array from the hybridization oven Vent the array with a clean pipette tip
13. on the magnetic stand used and the amount of aRNA in your sample x NOTE For maximum aRNA recovery mix well and ensure that the mixture is transparent all of the beads have been captured before proceeding Carefully aspirate and discard the supernatant without disturbing the magnetic beads then remove the plate from the magnetic stand 5 Bead Washing IMPORTANT Make sure that ethanol has been added to the bottle of aRNA Wash Solution Concentrate before using it Add 100 uL aRNA Wash Solution to each sample and shake at moderate speed for 1 minute setting 7 on the Lab Line Titer Plate Shaker NOTE The RNA Binding Beads may not fully disperse during this step this is expected and will not affect RNA purity or yield Move the plate to a magnetic stand and capture the RNA Binding Beads as in the previous step Carefully aspirate and discard the supernatant without disturbing the RNA Binding Beads and remove the plate from the magnetic stand 20 GeneChip 3 IVT Express Kit User Manual D Repeat Step A through Step C to wash a second time with 100 uL of aRNA Wash Solution E Move the plate to a shaker and shake the plate dry vigorously for 1 minute to evaporate residual ethanol from the beads setting 10 on the Lab Line Titer Plate Shaker 6 aRNA Elution A Elute the purified aRNA from the RNA Binding Beads by adding 50 uL preheated 50 60 C aRNA Elution Solution to each sample
14. rRNA peaks potential degradation products to provide a picture of RNA degradation states Search for RIN at the following web site for further information www chem agilent com Figure 2 1 Example Agilent Bioanalyzer Electropherograms from three different total RNAs of varying integrity Panel A represents a highly intact total RNA RIN 9 2 panel B represents a moderately intact total RNA RIN 6 2 and panel C represents a degraded total RNA sample RIN 3 2 FU FU FU A RIN 92 B RQN 62 C RIN 3 2 120 400 25 200 500 1000 2000 4000 Int 25 200 500 1000 2000 4000 nt 25 200 500 1000 2000 4000 nt NOTE Total RNAs with lower RIN values may require increased input amounts to generate enough aRNA for hybridization to an array Denaturing agarose gel electrophoresis and nucleic acid staining can also be used to separate and visualize the major rRNA species When the RNA resolves into discrete rRNA bands i e no significant smearing below each band with the 28S rRNA band appearing approximately twice as intense as the 18S rRNA band then the mRNA in the sample is likely to be mostly full length The primary drawback to gel electrophoresis is that it requires microgram amounts of RNA Chapter 2 aRNA Amplification Protocol 11 Other Important Parameters Keep reaction incubation times precise and consistent The incubation times for the enzymatic reactions in the protocol w
15. the RiboGreen fluorescence based assay for RNA quantitation Invitrogen is a convenient and sensitive way to measure RNA concentration Follow the manufacturer s instructions for using RiboGreen Expected Yield The aRNA yield will depend on the amount and quality of poly A RNA in the input total RNA Since the proportion of poly A RNA in total RNA is affected by influences such as health of the organism and the organ from which it is isolated aRNA yield from equal amounts of total RNA may vary considerably Figure 3 1 shows yield data for aRNA produced with the kit from several different types of input RNA 22 GeneChip 3 IVT Express Kit User Manual Figure 3 1 Average aRNA Yield from a variety of total RNA samples 140 0 16 hour IVT 4 hour IVT 120 0 RNA Sample Key 100 0 Pancreas E Thyroid B MAOC B Brain 80 0 Bg Heart B HeLa Cell Line Average Total aRNA Yield ug H 60 0 40 0 J 0 0 t 25 ng 50 ng 100 ng 100 ng 250 ng 500 ng Total RNA Input Amount Optional Concentrate the purified aRNA If necessary concentrate the aRNA by vacuum centrifugation If the heater on the vacuum centrifuge has different settings use medium or low Check the progress of drying every 5 10 minutes and remove the sample from the concentrator when it reaches the desired volume Avoid drying aRNA samples to completion Chapter 3 Eval
16. AX Affymetrix User Manual GeneChip 3 IVT Express Kit For research use only Not for use in diagnostic procedures Trademarks Affymetrix Axiom Command Console DMET GeneAtlas GeneChip GeneChip compatible GeneTitan Genotyping Console NetAffx and Powered by Affymetrix are trademarks or registered trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered by one or more of the following patents U S Patent Nos 5 445 934 5 744 305 5 945 334 6 140 044 6 399 365 6 420 169 6 551 817 6 733 977 and D430 024 and other U S or foreign patents Products are manufactured and s
17. Affymetrix P N 901530 Components needed Nuclease free water u 1 3X Hybridization Solution A a 1 3X Hybridization Solution B a GeneChip HT 3 IVT Express Kit Components Affymetrix P N 901225 4 x 24 rxn or P N 901253 96 rxn Components needed Control Oligo B2 3 nM 20X Hybridization Controls Preparing the Hybridization Cocktail Master Mix NOTE Refer to Table4 3 for the Hybridization Cocktail Master Mix composition 1 Obtain a 15 mL BD Falcon Test Tube or a 50 mL centrifuge tube for larger volumes and label as Hyb Mix 2 Take 1 3X Hybridization Solution A and Solution B from the GeneTitan Hybridization Wash and Stain Kit and warm to room temperature on the bench A Vortex 3 Remove Control Oligo B2 and 20X Hybridization Controls from 20 C and thaw at room temperature A Vortex and spin 4 Make a Hybridization Cocktail Master Mix according Table 4 3 in a 15 mL Falcon tube Use a 2 0 mL Eppendorf tube for 16 Array Plates A Vortex well Chapter 4 Hybridization 31 Table 4 3 HT Array Plate Hybridization Cocktail Master Mix using the GeneTitan Hybridization Wash and Stain Kit for 3 IVT Arrays P N 901530 Volume per 16 Array Plate 24 Array Plate 96 Array Plate Array uL 3 nM B2 Oligo 2 uL 36 uL 60 uL 210 uL 20X BioB C D Cre controls 6 uL 108 uL 180 uL 630 uL 1 3X Hybridization Solution A 32 3 uL 581 4 uL 969 uL 3 391 5 uL 1 3X Hybri
18. NA Samples Poly A RNA Spike Final Concentration ratio of copy number lys 1 100 000 phe 1 50 000 thr 1 25 000 dap 1 6 667 The controls are then amplified and labeled together with the total RNA samples Examining the hybridization intensities of these controls on GeneChip arrays helps to monitor the labeling process independently from the quality of the starting RNA samples The Poly A RNA Control Stock and Poly A Control Dil Buffer are provided in the GeneChip IVT Express Kit to prepare the appropriate serial dilutions based on Table 2 5 This is a guideline when 50 100 250 or 500 ng of total RNA is used as starting material For starting sample amounts other than those listed here calculations are needed in order to perform the appropriate dilutions to arrive at the same proportionate final concentration of the spike in controls in the samples bu IMPORTANT Use non stick RNase free microfuge tubes to prepare all of the dilutions not included Dilution of Poly A RNA Control Stock Total RNA Serial Dilutions Volume of 4t Input Amount FSU 5 dit TANA Dili Fourth DIIGd dilution to add irst Dilution econd Dilution ird Dilution Fou ilution to total RNA 50 ng 1 20 1 50 1 50 1 20 2 uL 100 ng 1 20 1 50 1 50 1 10 2 uL 250 ng 1 20 1 50 1 50 1 4 2 uL 500 ng 1 20 1 50 1 50 1 2 2 uL Recommendation Avoid pipetting solutions less than 2 uL in volume to maintain precision and consistenc
19. ample of the reaction on an Agilent bioanalyzer using an Agilent RNA 6000 Nano Kit Figure 3 3 shows a typical result of such analysis Follow the manufacturer s instructions for this analysis The reaction should produce a distribution of 35 200 nt aRNA fragments with a peak at approximately 100 120 nt Chapter 3 Evaluation and Fragmentation of aRNA 25 Figure 3 3 Example Agilent Bioanalyzer Electropherogram of fragmented aRNA FUL Fragmented aRNA 144 124 Peak Size 100 120 nt 25 200 500 1000 2000 4000 nt 4 Use fragmented aRNA immediately or store frozen Use the fragmented aRNA immediately or store undiluted fragmented aRNA at 20 C or 70 C for longer term storage 26 GeneChip 3 IVT Express Kit User Manual Hybridization This chapter provides instruction for setting up hybridizations for both cartridge arrays as well as HT Array Plates processed on the GeneTitan Instrument Target Hybridization for Cartridge Arrays This section provides instruction for setting up cartridge array hybridizations using the GeneChip Hybridization Wash and Stain Kit 30 rxns For ordering information please refer to Table 1 2 on page 5 Table 4 1 lists the necessary amount of aRNA required for the specific probe array format used These preparations take into account that it is necessary to make extra hybridization cocktail due to a small loss of volume 10 20 uL during each hybridiza
20. anual
21. cubation centrifuge briefly 5 seconds to collect the double stranded cDNA at the bottom of the tube plate C Place on ice and immediately proceed to the IVT below or freeze at 20 C El TIP STOPPING POINT Samples can be stored overnight at 20 C at this point if desired Chapter 2 aRNA Amplification Protocol 17 In Vitro Transcription to Synthesize Labeled aRNA Components of the GeneChip IVT Express Kit box 2 are used for this step 1 Assembly of IVT Master Mix A At room temp prepare an IVT Master Mix in a nuclease free tube in the order listed in Table 2 9 Prepare master mix for all the samples in the experiment including 5 overage to cover pipetting error Table 2 9 IVT Master Mix for a single reaction Component Amount IVT Biotin Label 4 uL IVT Labeling Buffer 20 uL IVT Enzyme Mix 6 uL Total Volume 30 pL B Mix well by gently vortexing Centrifuge briefly 5 seconds to collect the mix at the bottom of the tube and place on ice C Transfer 30 uL of IVT Master Mix to each 30 uL double stranded cDNA sample Mix thoroughly by gently vortexing and centrifuge briefly to collect the reaction at the bottom of the tube plate D Once assembled place the reaction in the thermal cycler block 2 Incubation Incubate the IVT reaction for 4 or 16 hours at 40 C in a thermal cycler using the program for IVT Table 2 3 on page 12 The recommended incubation time 1s based o
22. dization Solution B 60 uL 1 080 uL 1 800 uL 6 300 uL Nuclease free Water 3 7 uL 66 6 uL 111 uL 388 5 uL Total Volume 104 pL 1 872 uL 3 120 pL 10 920 pL For 16 Array Plates make the Master Mix in a 2 0 mL Eppendorf tube and dispense directly to the PCR plate Please refer to Important note below m IMPORTANT It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Controls are heated to 65 C for 5 minutes to completely resuspend the aRNA before aliquoting 5 For 16 Array Plates aliquot 104 uL of Hybridization Cocktail Master Mix directly to a 96 well plate The PCR plate must be compatible with the thermocycler being used m Using an 8 tip multi channel pipette aliquot to columns 5 and 7 only 6 For 24 and 96 Array Plates pour the Hybridization Cocktail Master Mix into a low volume reservoir on the bench at room temperature u Aliquot 104 uL to a 96 well PCR plate using an 8 tip multi channel pipette The PCR plate must be compatible with the thermocycler being used For a 24 Array Plate aliquot to columns 5 7 and 9 only 7 Transfer 16 uL of labeled and fragmented target 6 ug to 104 uL of Hybridization Cocktail to create the Hybridization ready Sample Plate 8 Seal the plate vortex spin 9 Proceed to Hybridization Setup on page 32 32 GeneChip 3 IVT Express Kit User Manual Hybridization Setup This section describes the GeneTitan Setup protocol for HT A
23. e not included to insure thorough pre heating of the Elution Solution 1 Preparation of aRNA Binding Mix Lus IMPORTANT Prepare only the amount needed for all samples in the experiment plus 10 overage to cover pipetting error At room temperature assemble aRNA Binding Mix in a nuclease free tube for all the samples in the experiment following the instructions in Table 2 11 Table 2 11 aRNA Binding Mix Preparation Instructions for a single reaction Component Amount RNA Binding Beads 10 uL aRNA Binding Buffer Concentrate 50 uL Mix the RNA Binding Beads by vortexing before dispensing Chapter 2 aRNA Amplification Protocol 19 2 Addition of aRNA Binding Mix A B C Add 60 uL aRNA Binding Mix to each sample Transfer each sample to a well of a U Bottom Plate Mix by pipetting up and down several times 3 aRNA binding A B C Add 120 uL 100 ethanol to each sample Mix by pipetting up and down several times Gently shake for 2 2 minutes to thoroughly mix setting 4 on the Lab Line Titer Plate Shaker The aRNA in the sample will bind to the RNA Binding Beads during this incubation 4 RNA Binding Beads capture A Move the plate to a magnetic stand and capture the magnetic beads for 5 minutes When capture is complete the mixture becomes transparent and the RNA Binding Beads will form pellets against the magnets in the magnetic stand The exact capture time depends
24. ere optimized in conjunction with the kit reagents for maximum yield in each step adhere to them closely Use master mixes We strongly recommend preparing master mixes for each step ofthe GeneChip 3 IVT Express procedure This reduces the effects of pipetting error saves time and improves reproducibility The fill volumes in the kit allow for a 5 overage when making master mixes Mix each kit component before use u Mix enzyme solutions by gently flicking the tube a few times before adding them to master mixes n Thaw frozen reagents completely at room temperature then mix thoroughly by vortexing and place on ice Incubate reactions in a calibrated thermal cycler a We do not recommend using ordinary laboratory heat blocks water baths or hybridization ovens for any of the reaction incubations u The procedure is very sensitive to temperature therefore use a thermal cycler that has been calibrated according to the manufacturer s recommended schedule Variable or inaccurate incubation temperatures can negatively impact aRNA synthesis o Heated lids It is important that condensation does not form in the tubes during any ofthe incubations because it would change the reaction composition and can greatly reduce yield If possible set the lid temperature to match the block temperature Otherwise incubate all reactions with the heated lid on 7100 C Maintain procedural consistency Procedural consistency 1s v
25. ery important for amplification experiments Consider implementing a detailed procedural plan that will be used by everyone in the lab to maintain consistency This type of plan will minimize variation due to subtle procedural differences that can influence RNA amplification and may complicate gene expression studies The plan should include basic information such as the method of RNA isolation the amount of RNA to use in the procedure and how long to incubate the IVT reaction It should also address specifics that are not often included in protocols such as which tubes and thermal cycler to use for each step in the process Finally develop a consistent work flow For example standardize stopping points in the method The idea 1s to standardize all of the variables discussed in this section of the Instruction Manual and carefully follow all the protocol steps in order to maximize amplification consistency among samples 12 GeneChip 3 IVT Express Kit User Manual Equipment and Reagent Preparation Prepare aRNA Wash Solution u Add 100 ethanol ACS reagent grade or equivalent to the bottle labeled aRNA Wash Solution Concentrate as indicated on the label Mix well and mark the label to indicate that the ethanol was added This solution will be referred to as aRNA Wash Solution in these instructions Store at room temperature Program the Thermal Cycler Incubate all reactions in a thermal cycler We find it convenient to set up the therma
26. ion Time 50 250 ng 16 hours 100 500 ng 4 hours RNA Purity RNA quality is the single most important factor affecting how efficiently an RNA sample will be amplified using GeneChip 3 IVT Express Kit RNA samples should be free of contaminating proteins DNA and other cellular material as well as phenol ethanol and salts associated with RNA isolation procedures Impurities can lower the efficiency of reverse transcription and subsequently reduce the level of amplification 10 GeneChip 3 IVT Express Kit User Manual An effective measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm The ratio of A to A values should fall in the range of 1 7 2 1 RNA must be suspended in high quality water TE 10 mM Tris HCl 1 mM EDTA RNA Integrity The integrity of the RNA sample or the proportion that is full length is another important component of RNA quality Reverse transcribing partially degraded mRNAs will generate cDNAs that may lack portions of the transcripts that are interrogated by probes on the array RNA integrity can be evaluated by microfluidic analysis using the Agilent 2100 Bioanalyzer with an RNA LabChip Kit Primarily full length RNA will exhibit a ratio of 28S to 18S rRNA bands that approaches 2 1 Using a bioanalyzer the RIN RNA Integrity Number can be calculated to further evaluate RNA integrity The RIN a metric developed by Agilent includes information from both the rRNA bands and outside the
27. ization Wash and Stain Kit for 3 IVT Arrays Or equivalent 6 GeneChip 3 IVT Express Kit User Manual Lab Equipment and Supplies Table 1 3 Lab Equipment and Supplies Material Source P N Lab Equipment and Supplies Thermal Cycler with heated Lid multiple capable of holding 0 2 mL tubes for reaction incubations Vortex Mixer multiple Microcentrifuge with an adapter for the PCR strip tubes or multiple plates supplied with the kit Magnetic Stand for 96 well plates Ambion AM10050 96 well Magnetic Stand or AM10027 Magnetic Stand 96 Orbital shaker for 96 well plates multiple e g Barnstead Lab Line Titer Plate Shaker Vacuum Centrifuge Concentrator Optional Spectrophotometer NanoDrop ND 8000 e g NanoDrop ND 8000 UV Vis Spectrophotometer Technologies Reagents and apparatus for preparation and electrophoresis of agarose gels Optional Miscellaneous Supplies Pipette for 0 1 to 2 uL Rainin L 2 Pipette for 2 to 20 uL Rainin L 20 Pipette for 20 to 200 uL Rainin L 200 Pipette for 100 to 1000 uL Rainin L 1000 Sterile barrier RNase free Pipette Tips multiple Bioanalyzer Agilent Non stick RNase free microfuge tubes 0 5 mL Ambion N12350 Non stick RNase free microfuge tubes 1 5 mL Ambion 12450 Or equivalent Tips must be pointed not rounded for efficient use with the probe arrays Beveled pipette tips may cause damage to
28. l cycler programs for each incubation before starting the procedure The specifications for each incubation are shown in Table 2 3 Table 2 3 Thermal Cycler Programs for RNA Amplification Program or Method First Strand cDNA Synthesis 42 C for 2 hrs 4 C indefinite hold Second Strand cDNA Synthesis 16 C for 1 hr 65 C for 10 min 4 C indefinite hold IVT 40 Cfor4or 16 hrs 4 Cindefinite hold Fragmentation 94 C for 35 min 4 C indefinite hold Preparation of Poly A RNA Controls Components of the GeneChip IVT Express Kit box 2 are used for this step Designed specifically to provide exogenous positive controls to monitor the entire eukaryotic target labeling process a set of poly A RNA controls is supplied in the GeneChip 3 IVT Express Kit Each eukaryotic GeneChip probe array contains probe sets for several B subtilis genes that are absent in eukaryotic samples lys phe thr and dap These poly A RNA controls are in vitro synthesized and the polyadenylated transcripts for the B subtilis genes are premixed at staggered concentrations The concentrated Poly A Control Stock can be diluted with the Poly A Control Dil Buffer and spiked directly into RNA samples to achieve the final concentrations referred to as a ratio of copy number summarized below in Table 2 4 Table 2 5 Serial Chapter 2 aRNA Amplification Protocol 13 Table 2 4 Final Concentrations of Poly A RNA Controls when added to total R
29. n such as RNaseZap Troubleshooting Low Yield and Small Average aRNA Size Consider the following troubleshooting suggestions if the positive control reaction produced the expected results but amplification of your experimental samples results in less or smaller average 500 nt aRNA than expected Lower Than Expected Input RNA Concentration Take another A260 reading of your RNA sample or if it is available try using 100 250 ng of RNA in the amplification procedure impure RNA Samples RNA samples with significant amounts of contaminating DNA protein phenol ethanol or salts are reverse transcribed poorly and subsequently generate less aRNA than pure RNA samples Phenol extract and ethanol precipitate your RNA or use a commercially available RNA cleanup kit to further purify your RNA before reverse transcription RNA Integrity is Compromised RNA that is partially degraded generates cDNA that is relatively short This will reduce the average size of the aRNA population and subsequently reduce the yield of aRNA You can assess the integrity of an RNA sample by determining the size of the 18S and 28S rRNA bands and the relative abundance of 28S to 188 rRNA See RNA Integrity on page 10 for more information The mRNA Content of Your total RNA Sample is Lower Than Expected Different RNA samples contain different amounts of mRNA In healthy cells mRNA constitutes 1 10 of total cellular RNA Johnson 1974 Sambrook and Russell
30. n the amount of input RNA and is shown in Table 2 10 Table 2 10 Recommended IVT Incubation Times RNA Amount IVT Incubation Time 50 250 ng 16 hours 100 500 ng 4 hours NOTE Optimal RNA input amount and IVT incubation time are sample type dependent and should be determined empirically It is recommended to keep input amount and IVT incubation time consistent within a given experiment 18 GeneChip 3 IVT Express Kit User Manual 3 Place the aRNA on ice briefly or freeze immediately Place the reaction on ice and proceed to the aRNA purification step below or immediately freeze at 20 C for overnight storage El TIP STOPPING POINT The aRNA can be stored overnight at 20 C at this point if desired aRNA Purification Components of the GeneChip IVT Express Kit box 1 are used for this step After synthesis the aRNA is purified to remove enzymes salts and unincorporated nucleotides Photos of the aRNA purification process can be found in Appendix B on page 37 If a plate shaker other than the recommended Lab Line Titer Plate Shaker will be used approximate shaking speeds for each step can be found in Appendix C Shaker Speeds on page 39 Before Beginning the aRNA Purification Preheat the aRNA Elution Solution to 50 60 C for at least 10 minutes NOTE Aliquot the appropriate amount of aRNA Elution Solution 50 pL per sample plus 10 overage to a separate 1 5 mL RNase Free Tub
31. old under license from OGT under 5 700 637 and 6 054 270 Copyright 2008 2010 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 OvervieW MM 1 Assay OVENIEW TTE 2 Control RNA 3 44 d a Lah dl dissidents 2 Kit Contents and Storage Conditions 4 Materials ie 333444 kb de HIS EXCDePRICBRRMeSeH bee kd Ted 5 Required Reagents 5 Lab Equipment and Supplies 6 MPVS ECM ac ep Cr m 7 aRNA Amplification Protocol 9 Important Parameters for Successful Amplification 9 Equipment and Reagent Preparation 12 Preparation of Poly A RNA Controls 12 Reverse Transcription to Synthesize First Strand cDNA 15 Second Strand cDNA Synthesis 16 In Vitro Transcription to Synthesize Labeled aRNA 17 ARNA PUNITIGATION 2 4 222 ti ind gott mm pm 18 Evaluation and Fragmentation of aRNA 21 aRNA Quantitation and Expected Yield 21 Analysis of aRNA Size 23 Fragmentation of Labeled aRNA 24 HybridizZati N esu ss sa ak kak a kk lale Wl kalk RE kal da ee Res 27 Target Hybridization for Cartridge Arrays
32. rray Plates The reagent consumption per process on the GeneTitan Instrument for processing HT array plates is shown in Table 4 5 Table 4 4 The Minimum Volumes of Buffer and Rinse Required to Process on the GeneTitan Instrument Minimum Level in Bottle Amount Required for One Fluid Type Array Plate One Array Plate Two Array Plates Rinse 300 mL 450 mL 900 mL Wash A 920 mL 1 040 mL 2 000 mL Wash B 300 mL 450 mL 600 mL Table 4 5 Volumes Required to Process HT Array Plates per Run Number of Plates that can be Processed using the GeneTitan Hybridization Wash and Stain Kit Amount H Aliquot into Columns for 3 IVT Arrays P N 901530 Required for Reagent One Array 16 24 96 16 Format 24 Format 96 Format Plate Format Format Format Hybridization Target 90 pL well 5 amp 7 5 7 amp 9 All N A N A N A Wash A 920 mL 1 1 1 Wash B 300 mL 1 1 1 Stain 1 and 3 105 uL well 5 amp 7 5 7 amp 9 All 6 4 1 Stain 2 105 uL well 5 amp 7 5 7 amp 9 All 6 4 1 Array Holding Buffer 150 uL well All All All 6 4 1 Please use GeneTitan Wash Buffers A and B Module PN 901583 for ordering Wash A and B buffers for additional plates IMPORTANT The instrument must have a minimum of 450 mL of Wash B in the Wash B reservoir of the instrument for each HT array plate prior to starting Hyb Wash Stain and Scan process The waste bottle should be empty 1 Carefully transfer the en
33. the array septa and cause leakage Instruments Table 1 4 Instruments Chapter 1 Overview 7 Instruments Manufacturer P N GeneChip Hybridization Oven 640 Affymetrix 800138 110 v 800139 220 v GeneChip Fluidics Station 450 Affymetrix 00 0079 GeneChip Scanner 3000 or higher Affymetrix See www affymetrix com GeneTitan Instrument Affymetrix See www affymetrix com 8 GeneChip 3 IVT Express Kit User Manual aRNA Amplification Protocol Important Parameters for Successful Amplification Input RNA Quantity and IVT Reaction Incubation Time x NOTE The RNA volume must be x 5 pL lt 3 pL if poly A RNA controls are used Consider both the type and amount of sample RNA available and the amount of aRNA needed for your analysis when planning experiments using the GeneChip 3 IVT Express Kit Because mRNA content varies significantly with tissue type the optimal amount of total RNA input and IVT incubation time should be determined empirically for each experimental system The recommended input RNA amounts listed in Table 2 1 are based on using total RNA from HeLa cells use these recommendations as a starting point Table 2 2 shows the corresponding recommended IVT incubation times Table 2 1 Input RNA Limits Recommendations Amount Recommended 100 ng Minimum 50 ng Maximum 500 ng Table 2 2 Recommended IVT Incubation Times RNA Amount IVT Incubat
34. tion 1 Mix the following for each target scaling up volumes if necessary for hybridization to multiple probe arrays lt NOTE DMSO will solidify when stored at 2 8 C Please ensure that the reagent is completely thawed prior to use After the first use it is recommended to store DMSO at room temperature Table 4 1 Hybridization Cocktail for Single Probe Array Component Array Format 49 Standard 100 Midi 169 Mini Final Dilution 64 Format 400 Micro Fragmented and Labeled aRNA 12 5 ug 33 3 uL 10 pg 26 7 uL 5 jg 13 3 uL 0 05 ug uL Control Oligonucleotide B2 3 nM 4 2 uL 3 3 uL 1 7 uL 50 pM 20X Hybridization Controls 12 5 uL 10 uL 5 uL 1 5 5 25 and bioB bioC bioD cre 100 pM respectively 2X Hybridization Mix 125 uL 100 uL 50 uL 1X DMSO 25 uL 20 uL 10 uL 10 Nuclease free Water 50 uL 40 uL 20 uL Total Volume 250 pL 200 pL 100 pL Please refer to specific probe array package insert for information on array format 28 GeneChip 3 IVT Express Kit User Manual IMPORTANT It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Controls are heated to 65 C for 5 minutes to completely resuspend the aRNA before aliquoting 2 Equilibrate probe array to room temperature immediately before use x NOTE It is important to allow the arrays to equilibrate to room temperature completely Specifically if the rubber septa are
35. tire amount of hybridization ready sample to a Biorad Hardshell PCR Plate using a multi channel pipette Chapter 4 Hybridization 33 Denature the hybridization ready target in the Biorad Hardshell plate for 5 minutes at 95 C and cool it to 45 C for 5 minutes After denaturing the target spin the plate in a centrifuge for 5 minutes at 5 000 RPM at room temperature to collect any insoluble material from the hybridization mixture Carefully transfer 90 uL of the denatured and centrifuged hybridization target into a HT Hybridization Tray using a multichannel pipette Please follow the instructions provided in the GeneTitan instrument User Guide for Expression Array Plates P N 702933 to process HT array plates on the GeneTitan Instrument 34 GeneChip 3 IVT Express Kit User Manual Troubleshooting Positive Control Reaction Control RNA Amplification Instructions To verify that the process is working as expected a Control RNA sample isolated from HeLa cells 1s provided with the kit 1 Dilute 2 uL of the Control RNA into 18 uL of Nuclease free Water 2 Use luL ofthe diluted Control RNA 100 ng follow the protocol starting at Reverse Transcription to Synthesize First Strand cDNA on page 15 3 At In Vitro Transcription to Synthesize Labeled aRNA on page 17 use a 16 hour incubation for the IVT reaction 4 Continue with the procedure for making biotin modified aRNA through aRNA Purification on page 18 Expected Results
36. uation and Fragmentation of aRNA 23 Analysis of aRNA Size The size distribution of aRNA can be evaluated using an Agilent 2100 bioanalyzer with the Agilent RNA 6000 Nano Kit P N 5067 1511 or by conventional denaturing agarose gel analysis The bioanalyzer can provide a fast and accurate size distribution profile of aRNA samples but aRNA yield should be determined by UV absorbance or RiboGreen analysis To analyze aRNA size using a bioanalyzer follow the manufacturer s instructions for running the assay using purified aRNA Expected aRNA Size We recommend analyzing aRNA size distribution using an Agilent bioanalyzer and RNA 6000 Nano Kit loaded with 300 ng of aRNA per well The expected aRNA profile is a distribution of sizes from 250 5500 nt with most of the aRNA between 600 1200 nt Average aRNA size may vary slightly depending on RNA quality and total RNA input amount Figure 3 2 Example Agilent Bioanalyzer Electropherogram of un fragmented aRNA generated from 50 ng of HeLa total RNA FU 404 Unfragmented aRNA 351 304 254 204 25 200 500 1000 2000 4000 nt NOTE Please refer to Chapter 4 for the amount of aRNA required for one array hybridization experiment The amount varies depending on the array format Please refer to the specific probe array package insert for information on the array format 24 GeneChip 3 IVT Express Kit User Manual Fragmentation of Labeled aRNA
37. use The kit is based upon linear RNA amplification and employs T7 in vitro transcription technology Also known as the Eberwine or reverse transcription IVT RT IVT method this process 1s considered the gold standard for target preparation for gene expression analysis RT IVT was experimentally validated using TaqMan RT PCR MAQC Consortium et al 2006 In the GeneChip 3 IVT Express Protocol total RNA undergoes reverse transcription to synthesize first strand cDNA This cDNA is then converted into a double stranded DNA template for transcription n vitro transcription synthesizes aRNA and incorporates a biotin congugated nucleotide CRNA is also known as amplified RNA or aRNA The aRNA is then purified to remove unincorporated NTPs salts enzymes and inorganic phosphate Fragmentation of the biotin labeled aRNA prepares the sample for hybridization onto GeneChip 3 expression arrays 2 GeneChip 3 IVT Express Kit User Manual Assay Overview The GeneChip 3 IVT Express Kit aRNA amplification procedure is depicted in Figure 1 1 Control RNA Reverse Transcription to Synthesize First Strand cDNA is primed with T7 oligo dT primer to synthesize cDNA containing a T7 promoter sequence Second Strand cDNA Synthesis converts the single stranded cDNA into a double stranded DNA dsDNA template for transcription The reaction employs DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA
38. y when preparing the dilutions 14 GeneChip 3 IVT Express Kit User Manual For example to prepare the poly A RNA dilutions for 100 ng of total RNA 1 Add 2 uL of the Poly A Control Stock to 38 uL of Poly A Control Dil Buffer for the first dilution 1 20 Mix thoroughly and spin down to collect the liquid at the bottom of the tube Add 2 uL of the First Dilution to 98 uL of Poly A Control Dil Buffer to prepare the Second Dilution 1 50 Mix thoroughly and spin down to collect the liquid at the bottom of the tube Add 2 uL of the Second Dilution to 98 uL of Poly A Control Dil Buffer to prepare the Third Dilution 1 50 Mix thoroughly and spin down to collect the liquid at the bottom of the tube Add 2 uL of the Third Dilution to 18 uL of Poly A Control Dil Buffer to prepare the Fourth Dilution 1 10 Mix thoroughly and spin down to collect the liquid at the bottom of the tube Add 2 uL of this Fourth Dilution to 100 ng of total RNA 13 NOTE The first dilution of the poly A RNA controls can be stored up to six weeks in a non frost free freezer at 20 C and frozen thawed up to eight times Table 2 6 Total RNA Poly A RNA Control Mixture Component Volume Total RNA Sample 50 500 ng variable Diluted Poly A RNA Controls 2 uL Fourth Dilution Nuclease free Water variable Total Volume 5 uL Chapter 2 aRNA Amplification Protocol 15 Reverse Transcription to Synthesize First Strand cDNA
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