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WT Plus Reagent Kit Assay Manual

1. Purify 2nd Cyde Single Stranded CDNA 2 0 e 22 Assess Single Stranded cDNA Yield and Size Distribution 0 000000000000 23 Fragment and Label Single Stranded CDNA 00 nee 24 WT Array Hybridization Rhone Rh ERR RRRERPEARGRR AS 26 Cartridge Array Hybridization on the GeneChip Instrument 0000005 26 Array Strips Hybridization on the GeneAtlas Instrument oaan asana aaa 30 Array Plates Hybridization on the GeneTitan Instrument n sasaaa 000 eee 41 Gel ShiTtASSaV saos Oe ca Xa 48 CR SOLAR RICE RD RRS ACCADE 45 Troubleshooting suse ordeum aem x RR pon Ro kook Rae RRR 47 References cz x ck da Snare a de hog a RASA SE quan Euge Adee eed ae te ee Dus 47 Revision History oe duas peche eg SEES TRE sq Rer ePRESESEE EE E EE 48 Technical Support 4 Technical Support Affymetrix Inc 3420 Central Expressway Santa Clara CA 95051 USA www affymetrix com Email supportGaffymetrix com Tel 1 888 362 2447 1 888 DNA CHIP Fax 1 408 731 5441 Affymetrix UK Ltd Voyager Mercury Park Wycombe Lane Wooburn Green High Wycombe HP10 OHH United Kingdom www affymetrix com Email supporteurope affymetrix com UK 0800 328 0056 France 08 00 91 95 05 Germany 01803 001334 Italy 800 91 59 74 Other 44 1628 552550 Fax 44 0 1628 552598 Affymetrix Japan K K ORIX Hamamatsucho Bldg 7F 1 24 8 Hamamatsucho Minato ku Tokyo 105 0013 Japan Email supportjapan affymetrix com Tel 81
2. WT PLUS Reagent Kit Contents and Storage 10 Reaction Kit 30 Reaction Kit Component for manual use for manual use Storage P N 902280 P N 902281 WT Amplification Kit Module 1 First Strand Enzyme 11 uL 50 uL 20 C First Strand Buffer 44 uL 160 uL 20 C Second Strand Enzyme 22 uL 70 uL 20 C Second Strand Buffer 198 uL 600 uL 20 C IVT Enzyme 66 uL 210 uL 20 C IVT Buffer 264 uL 800 uL 20 C Control RNA 1 mg mL HeLa total RNA 5 uL 5 uL 20 C 2nd Cycle Primers 44 uL 180 uL 20 C 2nd Cycle ss cDNA Enzyme 44 uL 140 uL 20 C 2nd Cycle ss cDNA Buffer 88 uL 290 uL 20 C RNase H 44 uL 180 uL 20 C Nuclease free Water 2x1 0 mL 4x1 0 mL any temp WT Amplification Kit Module 2 Purification Beads 2 2 mL 6 6 mL act GeneChip Poly A RNA Control Kit Poly A Control Stock 16 uL 16 uL 20 C Poly A Control Dil Buffer 3 8 mL 3 8 mL 20 C GeneChip WT Terminal Labeling Kit 10X cDNA Fragmentation Buffer 48 uL 213 uL 20 C UDG 10 U uL 10 uL 49 uL 20 C APE 1 1 000 U uL 10 uL 49 uL 20 C 5X TdT Buffer 120 uL 475 uL 20 C TdT 30 U uL 20 uL 99 uL 20 C DNA Labeling Reagent 5 mM 10 uL 49 uL 20 C RNase free Water 825 uL 2 x 825 uL any temp GeneChip Expression 3 Amplification Reagents Hybridization Control Kit 20X Hybridization Controls 450 uL 450 uL 20 C 3 nM Control Oligo B2 150 uL 150 uL 20 C equivalent 89 Store the Nuclea
3. 100 mL ofa 1X solution of SYBR Gold for staining SYBR Gold may be diluted in 1X TBE running buffer or water NOTE SYBR Gold is light sensitive Therefore use caution and shield the staining solution from light Prepare a new batch of stain at least once a week G After the gel is complete break open cartridge and stain the gel in 1X SYBR Gold for 10 min at room temperature Place the gel on a UV light box and image using the appropriate filter for SYBR Gold The absence of a shift pattern indicates poor biotin labeling The problem should be addressed before proceeding to the hybridization step Troubleshooting Observation Possible Cause Solution The positive control sample and your total RNA sample yield low levels of amplified cRNA product or low levels of appropriately sized cRNA product Incubation temperatures are incorrect or inaccurate Calibrate your thermal cycler Condensation formed in the tubes during the incubations Check that the heated lid is working correctly and is set to the appropriate temperature cRNA purification is not performed properly Perform the purification as described in this manual Pipettes tubes and or equipment are contaminated with nucleases Remove RNases and DNases from surfaces using RNase decontamination solution The positive control sample produces expected results but your total RNA sample results in low levels of amplified
4. 130 uL 80 uL C Remove the pipet tip from the upper right septum of the array Cover both septa with 1 2 Tough Spots to minimize evaporation and or prevent leaks D Place the arrays into hybridization oven trays Load the trays into the hybridization oven NOTE Ensure that the bubble inside the hybridization chamber floats freely upon rotation to allow the hybridization cocktail to make contact with all portions of the array E Incubate with rotation at 60 rpm for 16 hr at 45 C NOTE During the latter part of the 16 hr hybridization prepare reagents for the washing and staining steps required immediately after completion of hybridization Chapter 3 WT Array Hybridization 29 Wash and Stain For additional information about washing staining and scanning please refer to the Affymetrix GeneChip Fluidics Station 450 User s Guide AGCC P N 08 0295 the GeneChip Expression Wash Stain and Scan User Manual for Cartridge Arrays PN 702731 and the Affymetrix GeneChip Command Console User Manual P N 702569 Scan 1 2 Remove the arrays from the oven Remove the Tough Spots from the arrays Extract the hybridization cocktail mix from each array Optional Transfer it to a new tube or well of a 96 well plate in order to save the hybridization cocktail mix Store on ice during the procedure or at 20 C for long term storage Fill each array completely with Wash Buffer A Allow the arrays to equ
5. 2 1 on page 11 B Immediately after the incubation centrifuge briefly to collect the hydrolyzed 2nd cycle ss cDNA at the bottom of the tube or well C Place the samples on ice and proceed immediately to the next step Add Nuclease free Water to each hydrolyzed 2nd cycle ss cDNA sample A On ice add 11 uL of the Nuclease free Water to each 44 uL hydrolyzed 2nd cycle ss cDNA sample for a final reaction volume of 55 uL B Mix thoroughly by gently vortexing Centrifuge briefly to collect the reaction at the bottom of the tube or well C Place the sample on ice then proceed to Purify 2nd Cycle Single Stranded cDNA on page 22 or immediately freeze the samples at 20 C for storage TIP STOPPING POINT The hydrolyzed ss cDNA samples can be stored overnight at 20 C Chapter 2 Protocol 22 Purify 2nd Cycle Single Stranded cDNA After hydrolysis the 2nd cycle single stranded cDNA is purified to remove enzymes salts and unincorporated dNTPs This step prepares the cDNA for fragmentation and labeling Beginning the Single Stranded cDNA Purification E 1 B A IMPORTANT a Preheat the Nuclease free Water in a heat block or thermal cycler to 65 C for at least 10 min a Mix the Purification Beads thoroughly by vortexing before use to ensure that they are fully dispersed Transfer the appropriate amount of Purification Beads to a nuclease free tube or container and allow the Purification Beads to equilib
6. 3 6430 4020 Fax 81 3 6430 4021 For complete contact information and specific regional support contact information please go to www affymetrix com browse contactUs jsp WT PLUS Reagent Kit Product Information Purpose of the Product Safety The WT PLUS Reagent Kit enables you to prepare RNA samples for whole transcriptome expression analysis with GeneChip Whole Transcript WT Expression Arrays The kit generates amplified and biotinylated sense strand DNA targets from total RNA without the need for an up front selection or enrichment step for mRNA The kit is optimized for use with GeneChip Sense Target ST Arrays The WT PLUS Reagent Kit uses a reverse transcription priming method that primes the entire length of RNA including both poly A and non poly A mRNA to provide complete and unbiased coverage of the transcriptome The kit is comprised of reagents and a protocol for preparing hybridization ready targets from 50 to 500 ng of total RNA Figure 1 1 WT PLUS Reagent is optimized to work with a wide range of samples including tissues cells cell lines and whole blood The total RNA samples can be used directly without removal of ribosomal or globin RNA prior to target preparation with WT PLUS Reagent n WARNING For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals CAUTION All chemicals should be considered as pot
7. 3 uL or less Table 2 5 See Prepare Poly A RNA Controls on page 12 for more information For example when performing the Control RNA reaction combine 2 uL of RNA 25 ng uL 2 uL of diluted Poly A Spike Controls and 1 uL of Nuclease free Water NOTE If you are adding Poly A Spike Controls to your RNA the volume of RNA must be 3 uL or less If necessary use a SpeedVac or ethanol precipitation to concentrate the RNA samples Chapter 2 Protocol 15 Table 2 5 Total RNA Poly A RNA Control Mixture Component Volume for One Reaction Total RNA Sample 50 500 ng variable Diluted Poly A RNA Controls 4 Dilution 2 uL Nuclease free Water variable Total Volume 5 pL Synthesize First Strand cDNA In this reverse transcription procedure total RNA is primed with primers containing a T7 promoter sequence The reaction synthesizes single stranded cDNA with T7 promoter sequence at the 5 end x NOTE Avoid pipetting solutions less than 2 pL in volume to maintain precision and consistency High concentration RNA samples should be pre diluted with Nuclease free Water before adding to first strand cDNA synthesis reaction 1 Prepare First Strand Master Mix A On ice prepare the First Strand Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the total RNA samples in the experiment Include 5 excess volume to correct for pipettin
8. 9 affymetrix Biology for a better world User Manual GeneChip WT PLUS Reagent Kit Manual Target Preparation for GeneChip Whole Transcript WT Expression Arrays For Research Use Only Not for use in diagnostic procedures P N 703174 Rev 2 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 PLUS Reagently set forth in the Affymetrix terms and conditions 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 Atfymetrix 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 and or sold under license from Oxford Gene Technology
9. GeneChip arrays a If frozen the Poly A Control Dil Buffer may take 15 to 20 min to thaw at room temperature A set of poly A RNA controls supplied by Affymetrix is designed specifically to provide exogenous positive controls to monitor the entire target preparation It should be added to the RNA prior to the First Strand cDNA Synthesis step 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 in Table 2 2 Table 2 2 Final concentrations of Poly A RNA Controls when added to total RNA 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 Chapter 2 Protocol 13 The Poly A RNA Control Stock and Poly A Control Dil Buffer are provid
10. U S Patent Nos 5 445 934 5 700 637 5 744 305 5 945 334 6 054 270 6 140 044 6 261 776 6 291 183 6 346 413 6 399 365 6 420 169 6 551 817 6 610 482 6 733 977 and EP 619 321 373 203 and other U S or foreign patents Copyright 2013 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Appendix A Appendix B Appendix C Techical SUpport 2 g DOES m d RR oras o DID uu e RR RP dee 4 WT PLUS Reagent Kit ct ccnccs ca ccure eee a ar RA er d fed ea ones Ree ErbR 5 Product IntormiatiOr o s a nat Ais gic addins aceite Sed haan do dado s RUE RA ud 5 EC A EET 5 Assay WOIKTOW 3 3 22 bleue oe baie o bbd d bo eb PP ted buen d hd Pad 6 Kit Contents and Storage 0 2 ee eee eee 7 Required Materials 22 ssi 2299959445 meee es xd gpa hl fee ed ook ee eee edges 8 Protocol 2c ctacsehcbavadepncnretadasenesanhscwaneesankecevaeas 10 Procedural NOtes uso cee eee cec a Te cide ded erede Rene deed amp euler eens N 10 Prepar Control RNA 2 ate cbincbe Stata thoi oot eibi ed td heh tos 12 Prepare Total RNA EP Cc 13 Syrithesize First Strand CDNA scrobe eR CE EET GR XE REG d Recreo EESAN 15 Synthesize Second Strand CDNA 0 2 ns 16 Synthesize cRNA by In Vitro Transcription 0 0 0 0 2 00 cee 17 Purify cRNA i i eaa las 18 Assess CRNA Yield and Size Distribution 00 0000 n 19 Synthesize 2nd Cycle Single Stranded CDNA 0 ee 20 Hydrolyze RNA Using RNase H siete prp a OO RR boe eS 21
11. WT Hybridization Master Mix amp Cocktail A Heat the 20X Hybridization Controls for 5 min at 65 C in a thermal cycler using the Hybridization Control program that is shown in Table 2 1 on page 11 B Atroom temperature prepare the Hybridization Master Mix in a nuclease free tube Combine the appropriate amount of components in the sequence shown in the table below Prepare the master mix for all the fragmented and biotin labeled ss cDNA samples in the experiment f NOTE The 5X WT Hyb Add 1 solution is very viscous pipet slowly to ensure addition of the correct volume Mix well Vortex and centrifuge briefly 5 sec to collect liquid at the contents of the tube Table 3 7 Hybridization Master Mix Component Volume for 16 Array 24 Array 96 Array Final Concentration One Array Plate Plate Plate 5X WT Hyb Add 1 24 uL 422 4 uL 633 6 uL 2 534 4 uL 1X Control Oligo B2 3 nM 1 2 uL 21 1 uL 31 7 uL 126 7 uL 30 pM 20X Eukaryotic Hybridization Controls 6 uL 105 6 uL 158 4 uL 633 6 uL 1 5 5 25 and 100 pM bioB bioC bioD cre respectively 15X WT Hyb Add 4 8 uL 140 8 uL 211 2 uL 844 8 uL 1X Total Volume 39 2 uL 689 9 uL 1 034 90L 4 139 5 pL Includes 10 overage to cover pipetting error C Mix thoroughly by gently vortexing Centrifuge briefly to collect the mix and proceed immediately to the next step 3 Prepare Hybridization Cocktail A Atroom temperature prepare the Hybridization
12. cCRNA cDNA product The input total RNA concentration is lower than expected Repeat the A reading of your RNA sample Use 100 to 200ng of total RNA in the First Strand cDNA Synthesis procedure Your input RNA contains contaminating DNA protein phenol ethanol or salts causing inefficient reverse transcription Phenol extract and ethanol precipitate your total RNA The positive control sample produces expected results but your total RNA sample results in low levels of appropriately sized cRNA cDNA product The total RNA integrity is partially degraded thereby generating short cDNA fragments Assess the integrity of your total RNA sample by determining the size of the 18S and 28S rRNA bands and the relative abundance of 28S to 18S rRNA Refer to Evaluate RNA Integrity on page 13 The mRNA content of your total RNA sample is lower than expected Verify the mRNA content of your total RNA Note In healthy cells mRNA constitutes 1 to 1096 of total cellular RNA Johnson 1974 Sambrook and Russel 2001 References Johnson L F Abelson H T Green H and S Penman 1974 Cell 1 95 100 Sambrook J and D W Russel 2001 Extraction purification and analysis of mRNA from eukaryotic cells In Molecular cloning a laboratory manual third edition Vol 1 Cold Spring Harbor New York Cold Spring Harbor Press Van Gelder R N von Xastrow M E Yool E et al 1990 Proc Nat
13. collect the reaction at the bottom of the tube or well then proceed immediately to the next step 7 Incubate for 1 hr at 37 C then for 10 min at 70 C then for at least 2 min at 4 C A Incubate the labeling reaction in a thermal cycler using the Labeling program that is shown in Table 2 1 on page 11 Immediately after the incubation centrifuge briefly to collect the fragmented and labeled ss cDNA at the bottom of the tube or well Place the sample on ice then proceed to Chapter 3 WT Array Hybridization on page 26 or immediately freeze the samples at 20 C for storage 8 Optional Remove 2 uL of each fragmented and labeled ss cDNA sample for Gel shift analysis as described in Appendix A Gel Shift Assay on page 45 to assess the fragmentation and labeling efficiency E TIP STOPPING POINT The fragmented and labeled ss cDNA samples can be stored overnight at 20 C For long term storage at 20 C we recommend to store the samples as unfragmented and unlabeled ss cDNA WT Array Hybridization Cartridge Array Hybridization on the GeneChip Instrument This section provides instruction for setting up hybridizations for cartridge arrays Please refer to Affymetrix GeneChip Fluidics Station 450 User s Guide AGCC P N 08 0295 the GeneChip Expression Wash Stain and Scan User Manual for Cartridge Arrays PN 702731 and the Affymetrix GeneChip Command Console User Manual P N 702569 for further detai
14. in the upper right corner A confirmation message box appears Figure 3 18 Chapter 3 WT Array Hybridization 40 Figure 3 18 Confirmation Message A Are you sure you want to stop Click Yes to complete hybridization It is important to remove the hybridization tray from the Hybridization Station after the timer has completed the countdown as the Hybridization Station does not shut down when the hybridization is complete Save the remaining hybridization cocktail in 20 C for future use Immediately proceed to the GeneAtlas Wash Stain and Scan protocol Please refer to GeneAtlas System User s Guide P N 08 0306 for further detail Chapter 3 WT Array Hybridization 41 Array Plates Hybridization on the GeneTitan Instrument This chapter outlines the basic steps involved in hybridizing array plate s on the GeneTitan Instrument The two major steps involved in array plate hybridization are a Target Hybridization Setup for Affymetrix Array Plates on page 41 a Processing WT Array Plates on the GeneTitan Instrument on page 43 Please refer to GeneTitanG Instrument User Guide for Expression Arrays Plates P N 702933 and Affymetrix GeneChip Command Console User s Guide P N 702569 for further detail Target Hybridization Setup for Affymetrix Array Plates Reagents and Materials Required a GeneTitan Hybridization Wash and Stain Kit for WT Array Plates Not supplied For ordering information ple
15. shown in Table 2 1 on page 11 B Immediately after the incubation centrifuge briefly to collect the fragmented ss cDNA at the bottom of the tube or well C Place the sample on ice then proceed immediately to the next step Chapter2 Protocol 25 4 Optional The fragmented ss cDNA sample can be used for size analysis using a Bioanalyzer Please see the Reagent Kit Guide that comes with the RNA 6000 Nano LabChip Kit for detailed instructions The range in peak size of the fragmented samples should be approximately 40 to 70 nt 5 On ice transfer 45 uL of the fragmented ss cDNA sample to each tube or well Prepare Labeling Master Mix A On ice prepare the Labeling Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the fragmented ss cDNA samples in the experiment Include 5 excess volume to correct for pipetting losses Table 2 11 Labeling Master Mix Component Volume for One Reaction 5X TdT Buffer 12 uL DNA Labeling Reagent 5 mM Tul TdT 30 U uL 2 uL Total Volume 15 pL B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube then proceed immediately to the next step C On ice transfer 15 uL of the Labeling Master Mix to each 45 uL fragmented ss cDNA sample for a final reaction volume of 60 uL D Mix thoroughly by gently vortexing the tube Centrifuge briefly to
16. the GeneAtlas System The two major steps involved in array strip hybridization are a Target Hybridization Setup for Affymetrix Array Strips on page 30 a GeneAtlas Software Setup on page 35 1x NOTE If you are using a hybridization ready sample or re hybridizing previously made hybridization cocktail continue the protocol from Step 5 on page 32 bu IMPORTANT Before preparing hybridization ready samples register samples as described in GeneAtlas Software Setup on page 35 Please refer to GeneAtlas System User s Guide P N 08 0306 for further detail Target Hybridization Setup for Affymetrix Array Strips Reagents and Materials Required GeneAtlas Hybridization Wash and Stain Kit for WT Array Strips Not supplied For ordering information please refer to Table 1 4 on page 9 or the Affymetrix website o 5X WT Hyb Add 1 o 15X WT Hyb Add 4 n 2 5X WT Hyb Add 6 o Stain Cocktail 1 o Stain Cocktail 2 n Array Holding Buffer o Wash Buffer A u Wash Buffer B a GeneChip Hybridization Control Kit o 20X Eukaryotic Hybridization Controls bioB bioC bioD cre o Control Oligonucleotide B2 3 nM a Affymetrix Array Strip and consumables Not supplied u Affymetrix WT Array Strip s n 1 hybridization tray per array strip Procedure x NOTE The WT Hyb Add reagent names were created to match the order in which reagents are added For example WT Hyb Add 1 is the first component added during preparation of
17. the final wash solution D Air dry on the magnetic stand for 5 min until no liquid is visible yet the pellet appears shiny Additional time may be required Do not over dry the beads as this will reduce the elution efficiency The bead surface will appear dull and may have surface cracks when it is over dry 3 Elute cRNA A Remove the plate from the magnetic stand Add to each sample 27 uL of the preheated 65 C Nuclease free Water and incubate for 1 min Mix well by pipetting up and down 10 times Move the plate to the magnetic stand for 5 min to capture the Purification Beads Transfer the supernatant which contains the eluted cRNA to a nuclease free tube mon u Place the purified cRNA samples on ice then proceed to Assess cRNA Yield and Size Distribution or immediately freeze the samples at 20 C for storage NOTE a Minimal bead carryover will not inhibit subsequent enzymatic reactions a It may be difficult to resuspend magnetic particles and aspirate purified CRNA when the cRNA is very concentrated To elute the sample with high concentration cRNA add an additional 10 30 uL of the preheated Nuclease free Water to the well incubate for 1 min and proceed to Step 3B EH TIP STOPPING POINT The purified cRNA samples can be stored overnight at 20 C For long term storage store samples at 80 C and keep the number of freeze thaw cycles to 3 or less to ensure cRNA integrity Assess cRNA Yiel
18. to collect the reaction at the bottom of the tube or well then proceed immediately to the next step 2 Incubate for 1 hr at 16 C then for 10 min at 65 C then for at least 2 min at 4 C A Incubate the second strand synthesis reaction in a thermal cycler using the Second Strand cDNA Synthesis program that is shown in Table 2 1 on page 11 Lu IMPORTANT Disable the heated lid of the thermal cycler or keep the lid off during the Second Strand cDNA Synthesis B Immediately after the incubation centrifuge briefly to collect the second strand cDNA at the bottom of the tube or well C Place the sample on ice then proceed immediately to Synthesize cRNA by In Vitro Transcription on page 17 EH TIP When there is approximately 15 min left on the thermal cycler you may start reagent preparation for In Vitro Transcription Chapter 2 Protocol 17 Synthesize cRNA by In Vitro Transcription In this procedure antisense RNA complimentary RNA or cRNA is synthesized and amplified by in vitro transcription IVT of the second stranded cDNA template using T7 RNA polymerase This method of RNA sample preparation is based on the original T7 in vitro transcription technology known as the Eberwine or RT IVT method Van Gelder et al 1990 IMPORTANT a Transfer the second strand cDNA samples to room temperature for gt 5 min while preparing IVT Master Mix a After the IVT Buffer is thawed completely leave the IVT Buffer at r
19. 2 Dilution to 98 uL of Poly A Control Dil Buffer to prepare the 3 Dilution 1 50 Add 2 uL of the 3 Dilution to 18 uL of Poly A Control Dil Buffer to prepare the 4 Dilution 1 10 Add 2 uL of this 4 Dilution to 100 ng of total RNA The final volume of total RNA with the diluted Poly A controls should not exceed 5 uL EH TIP The first dilution of the Poly A RNA controls can be stored up to 6 weeks in a non frost free freezer at 20 C and frozen thawed up to eight times Label the storage tube with the expiration date for future reference Prepare Total RNA Evaluate RNA Quality RNA quality affects how efficiently an RNA sample is amplified using this kit High quality RNA is free of contaminating proteins DNA phenol ethanol and salts To evaluate RNA quality determine its A Aso ratio RNA of acceptable quality is in the range of 1 7 to 2 1 Evaluate RNA Integrity The integrity of the RNA sample or the proportion that is full length is an important component of RNA quality Reverse transcribing partially degraded mRNA may generate cDNA that lacks parts of the coding region Chapter2 Protocol 14 Two methods to evaluate RNA integrity are a Microfluidic analysis using the Agilent 2100 Bioanalyzer with an RNA LabChip Kit or equivalent instrument Denaturing agarose gel electrophoresis With microfluidic analysis you use the RNA Integrity Number RIN to evaluate RNA integrity For more information on how
20. 2 96 rxns Nuclease free aerosol barrier tips Major Laboratory Supplier Nuclease free 1 5 and 0 2 mL tubes or plates Major Laboratory Supplier Nuclease free 15 mL tubes or containers Major Laboratory Supplier Optional materials for Gel Shift assay refer to Appendix A Gel Shift Assay on page 45 Optional RNA 6000 Nano Kit Agilent Technologies Inc P N 5067 1511 or equivalent DNA and RNA sizing reagents Tough Spots Major Laboratory Supplier 100 Ethanol Molecular Biology grade or equivalent Major Laboratory Supplier Nuclease free Water for preparing 80 ethanol wash solution Affymetrix P N 71786 or major laboratory supplier Optional 96 well plate sealing film Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all relevant precautions Major Laboratory Supplier Protocol Procedural Notes Implement a Plan to Maintain Procedural Consistency To minimize sample to sample variation that is caused by subtle procedural differences in gene expression assays consider implementing a detailed procedural plan The plan standardizes the variables in the procedure and should include the Method of RNA isolation Amount of input RNA that is used for each tissue type a RNA purity and integrity a Equipment Preparation Workflow stopping points a Reagent Preparation Equipment Preparation Recommended Thermal Cy
21. 5 5 ug of single stranded cDNA is required for fragmentation and labeling 1 Prepare 5 5 ug of ss cDNA On ice prepare 176 ng uL ss cDNA This is equal to 5 5 ug ss cDNA in a volume of 31 2 uL If necessary use Nuclease free Water to bring the ss cDNA sample to 31 2 uL 2 Prepare Fragmentation Master Mix A On ice prepare the Fragmentation Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the ss cDNA samples in the experiment Include 5 excess volume to correct for pipetting losses Table 2 10 Fragmentation Master Mix Component Volume for One Reaction Nuclease free Water 10 uL 10X cDNA Fragmentation Buffer 4 8 uL UDG 10 U uL 1 uL APE 1 1 000 U uL 1 uL Total Volume 16 8 uL B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube then proceed immediately to the next step C On ice transfer 16 8 uL of the Fragmentation Master Mix to each 31 2 uL purified ss cDNA sample for a final reaction volume of 48 uL D Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the reaction at the bottom of the tube or well then proceed immediately to the next step 3 Incubate for 1 hr at 37 C then for 2 min at 93 C then for at least 2 min at 4 C A Incubate the fragmentation reaction in a thermal cycler using the Fragmentation program that is
22. Cocktail in the order as shown in Table 3 8 for all samples Table 3 8 Hybridization Cocktail for a Single Array Component Volume for One Array Final Concentration Hybridization Master Mix 39 2 uL Fragmented and labeled ss cDNA 32 8 uL 23 ng uL 2 5X WT Hyb Add 6 48 uL 1X Total Volume 120 pL B If you are using a plate seal vortex and centrifuge briefly 5 sec to collect liquid at the bottom ofthe well If you are using 1 5 mL tubes vortex and centrifuge briefly 5 sec to collect contents of the tube C Incubate the hybridization cocktail reaction for 5 min at 99 C tubes or 95 C plates then for 5 min at 45 C in a thermal cycler using the Hybridization Cocktail program that is shown in Table 2 1 on page 11 D After the incubation centrifuge briefly to collect contents of the tube or well and proceed immediately to the next step E Place 90 uL of the centrifuged supernatant hybridization cocktail as indicated into the appropriate well of the hybridization tray F Proceed to Hybridization Setup on page 43 Chapter 3 WT Array Hybridization 43 Hybridization Setup This section describes the GeneTitan Setup protocol for WT Array Plates The reagent consumption per process on the GeneTitan Instrument for processing WT Array Plates is shown in Table 3 10 Table 3 9 The Minimum Volumes of Buffer and Rinse Required to Process on the GeneTitan Instrument Minimum Level in Bo
23. P N 00 0213 International GeneChip AutoLoader with External Barcode Reader Affymetrix P N 00 0090 GCS 3000 7G S N 501 P N 00 0129 GCS 3000 7G S N 502 GeneAtlas System for Array Strips GeneAtlas Workstation Affymetrix P N 90 0894 GeneAtlas Hybridization Station Affymetrix P N 00 0380 115VAC P N 00 0381 230VAC GeneAtlas Fluidics Station Affymetrix P N 00 0377 GeneAtlas Imaging Station Affymetrix P N 00 0376 GeneAtlas Barcode Scanner Affymetrix P N 74 0015 Table 1 3 Instruments Required for Array Processing Continued Chapter 1 WT PLUS Reagent Kit Reagents and Supplies Table 1 4 Additional Reagents and Supplies Required Instruments Supplier Part Number GeneTitan System for Array Plates GeneTitan MC Instrument NA Japan includes 110v UPS Affymetrix P N 00 0372 GeneTitan MC Instrument Int l includes 220v UPS Affymetrix P N 00 0373 GeneTitan Instrument NA Japan includes 110v UPS Affymetrix P N 00 0360 GeneTitan Instrument Int l Includes 220v UPS Affymetrix P N 00 0363 Item Supplier 96 well round bottom microtiter plate Costar P N 3795 or equivalent GeneChip Hybridization Wash and Stain Kit Affymetrix P N 900720 30 rxns GeneAtlas Hybridization Wash and Stain Kit for WT Array Strips Affymetrix P N 901667 60 rxns GeneTitan Hybridization Wash and Stain Kit for WT Array Plates Affymetrix P N 90162
24. antly reduce cRNA yields Holding the First Strand cDNA Synthesis reaction at 4 C for longer than 10 min may significantly reduce cRNA yields E TIP When there is approximately 15 min left on the thermal cycler you may start reagent preparation for Second Strand cDNA Synthesis Synthesize Second Strand cDNA In this procedure single stranded cDNA is converted to double stranded cDNA which acts as a template for in vitro transcription The reaction uses DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA Lu IMPORTANT Pre cool thermal cycler block to 16 C 1 Prepare Second Strand Master Mix A On ice prepare the Second Strand Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the first strand cDNA samples in the experiment Include 5 excess volume to correct for pipetting losses Table 2 7 Second Strand Master Mix Component Volume for One Reaction Second Strand Buffer 18 uL Second Strand Enzyme 2 uL Total Volume 20 pL B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube and proceed immediately to the next step C On ice transfer 20 uL of the Second Strand Master Mix to each 10 uL first strand cDNA sample for a final reaction volume of 30 uL D Mix thoroughly by gently vortexing the tube Centrifuge briefly
25. ase refer to Table 1 4 on page 9 or the Affymetrix website o 5X WT Hyb Add 1 o 15X WT Hyb Add 4 o 2 5X WT Hyb Add 6 o Stain Cocktail 1 amp 3 o Stain Cocktail 2 n Array Holding Buffer o Wash Buffer A u Wash Buffer B a GeneChip Hybridization Control Kit o 20X Eukaryotic Hybridization Controls bioB bioC bioD cre o Control Oligonucleotide B2 3 nM a Affymetrix Array Plate and consumables Not supplied o Affymetrix WT Array Plate s Procedure f NOTE The WT Hyb Add reagent names were created to match the order in which reagents are added For example WT Hyb Add 4 is the fourth component added during preparation of the Hybridization Mix WT Hyb Add 2 3 and 5 are not used and are not part of the Hybridization Module 1 In preparation of the hybridization step prepare the following A Warm the following vials to room temperature on the bench a 5X WT Hyb Add 1 a 15X WT Hyb Add 4 a 2 5X WT Hyb Add 6 B Vortex and centrifuge briefly 5 sec to collect contents of the tube C Remove the following tubes from the GeneChip Hybridization Control Kit and thaw at room temperature Control Oligonucleotide B2 3 nM a 20X Eukaryotic Hybridization Controls D Vortex and centrifuge briefly 5 sec to collect liquid at the bottom of the tube E Keep the tubes of Control Oligonucleotide B2 3 nM and the tube of 20X Eukaryotic Hybridization Controls on ice Chapter 3 WT Array Hybridization 42 2 Prepare the
26. c stand to capture the Purification Beads When capture is complete after 5 min the mixture is transparent and the Purification Beads form pellets against the magnets in the magnetic stand The exact capture time depends on the magnetic stand that you use and the amount of ss cDNA generated by 2nd Cycle ss cDNA Synthesis Carefully aspirate and discard the supernatant without disturbing the Purification Beads Keep the plate on the magnetic stand 2 Wash the Purification Beads A While on the magnetic stand add 200 uL of 80 ethanol wash solution to each well and incubate for 30 sec Chapter 2 Protocol 23 B Slowly aspirate and discard the 80 ethanol wash solution without disturbing the Purification Beads C Repeat Step A and Step B twice for a total of 3 washes with 200 uL of 80 ethanol wash solution Completely remove the final wash solution D Air dry on the magnetic stand for 5 min until no liquid is visible yet the pellet appears shiny Additional time may be required Do not over dry the beads as this will reduce the elution efficiency The bead surface will appear dull and may have surface cracks when it is over dry 3 Elute ss cDNA A Remove the plate from the magnetic stand Add to each sample 30 uL of the preheated 65 C Nuclease free Water and incubate for 1 min Mix well by pipetting up and down 10 times Move the plate to the magnetic stand for 5 min to capture the Purification Beads Tran
27. cler Make sure that the heated cover of your thermal cycler either tracks the temperature of the thermal cycling block or supports specific temperature programming Program the Thermal Cycler Set the temperature for the heated lid to or near the required temperature for each step An alternate protocol may be used for thermal cyclers that lack a programmable heated lid This is not the preferred method Yields of cRNA may be greatly reduced if a heated lid is used during the Second Strand cDNA Synthesis or during the In Vitro Transcription cRNA Synthesis steps We recommend leaving the heated lid open during Second Strand cDNA Synthesis A small amount of condensation will form during the incubation This is expected and should not significantly decrease cRNA yields For In Vitro Transcription cRNA Synthesis we recommend incubating the reaction in a 40 C hybridization oven if a programmable heated lid thermal cycler is unavailable Incubation temperatures and times are critical for effective RNA amplification Use properly calibrated thermal cyclers and adhere closely to the incubation times NOTE Concentration fluctuations that are caused by condensation can affect yield Ensure that the heated lid feature of the thermal cycler is working properly Table 2 1 Thermal Cycler Programs Chapter 2 Protocol 11 Program Heated Alternate Step 1 Step 2 Step 3 Step 4 Volume Lid Temp Protocol First Stra
28. d and Size Distribution Expected cRNA Yield The cRNA yield depends on the amount and quality of non rRNA in the input total RNA Because the proportion of non rRNA in total RNA is affected by factors such as the health of the organism and the organ from which it is isolated cRNA yield from equal amounts of total RNA may vary considerably During development of this kit using a wide variety of tissue types 50 ng of input total RNA yielded 15 to 40 ug of cRNA For most tissue types the recommended 100 ng of input total RNA should provide gt 20 ug of cRNA Determine cRNA Yield by UV Absorbance Determine the concentration of a cRNA solution by measuring its absorbance at 260 nm Use Nuclease free Water as blank Affymetrix recommends using NanoDrop Spectrophotometers for convenience No dilutions or cuvettes are needed just use 1 5 uL of the cRNA sample directly Samples with cRNA concentrations greater than 3 000 ng uL should be diluted with Nuclease free Water before measurement and reaction setup Use the diluted cRNA as the input to prepare 15 ug cRNA in 2nd cycle cDNA synthesis reaction Alternatively determine the cRNA concentration by diluting an aliquot of the preparation in Nuclease free Water and reading the absorbance in a traditional spectrophotometer at 260 nm Calculate the concentration in pg mL using the equation shown below 1 A 40 ug RNA mL A X dilution factor x 40 ug RNA mL Chapter 2 Protocol 20 Optiona
29. d with Nuclease free Water before measurement and reaction setup Use the diluted cRNA as the input to prepare 15 ug of cRNA 2 Prepare cRNA and 2nd Cycle Primers Mix A On ice combine 24 uL of cRNA 15 ug 4 uL of 2nd Cycle Primers B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube then proceed immediately to the next step 3 Incubate for 5 min at 70 C then 5 min at 25 C then 2 min at 4 C A Incubate the cRNA Primers mix in a thermal cycler using the 2nd Cycle Primers cRNA Annealing program that is shown in Table 2 1 on page 11 B Immediately after the incubation centrifuge briefly to collect the cRNA Primers mix at the bottom of the tube or well C Place the mix on ice then proceed immediately to the next step 4 Prepare 2nd Cycle ss cDNA Master Mix A On ice prepare the 2nd Cycle ss cDNA Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the cRNA Primers samples in the experiment Include 5 excess volume to correct for pipetting losses Table 2 9 2nd Cycle ss cDNA Master Mix Component Volume for One Reaction 2nd Cycle ss cDNA Buffer 8 uL 2nd Cycle ss cDNA Enzyme 4 uL Total Volume 12 pL 5 Chapter2 Protocol 21 B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube and proceed imm
30. ed in the GeneChip Poly A RNA Control Kit to prepare the appropriate serial dilutions based on Table 2 3 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 Table 2 3 Serial Dilution of Poly A RNA Control Stock Total RNA Serial Dilutions Volume of 4 Dilution Input Amount TES IX TAE to Add to Total RNA 1st Dilution 2 4 Dilution 3 Dilution 4th Dilution 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 IMPORTANT Avoid pipetting solutions less than 2 pL in volume to maintain precision and consistency when preparing the dilutions a Use non stick nuclease free tubes to prepare all of the dilutions not included a After each step mix the Poly A Control dilutions thoroughly by gently vortexing followed by a quick centrifuge to collect contents of the tube For example to prepare the Poly A RNA dilutions for 100 ng of total RNA 1 uBWN Add 2 uL of the Poly A Control Stock to 38 uL of Poly A Control Dil Buffer for the 1 Dilution 1 20 Add 2 uL of the 1 Dilution to 98 uL of Poly A Control Dil Buffer to prepare the 274 Dilution 1 50 Add 2 uL of the
31. ediately to the next step C On ice transfer 12 uL of the 2nd Cycle ss cDNA Master Mix to each 28 uL cRNA 2nd Cycle Primers sample for a final reaction volume of 40 uL D Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the reaction at the bottom of the tube or well then proceed immediately to the next step Incubate for 10 min at 25 C then 90 min at 42 C then 10 min at 70 C then for at least 2 min at 4 C A Incubate the 2nd cycle synthesis reaction in a thermal cycler using the 2nd Cycle ss cDNA Synthesis program that is shown in Table 2 1 on page 11 B Immediately after the incubation centrifuge briefly to collect the 2nd cycle ss cDNA at the bottom of the tube or well C Place the sample on ice and proceed immediately to Hydrolyze RNA Using RNase H on page 21 Hydrolyze RNA Using RNase H In this procedure RNase H hydrolyzes the cRNA template leaving single stranded cDNA 1 Add RNase H to each 2nd cycle ss cDNA sample A On ice add 4 uL of the RNase H to each 40 uL 2nd cycle ss cDNA sample for a final reaction volume of 44 uL B Mix thoroughly by gently vortexing Centrifuge briefly to collect the reaction at the bottom of the tube or well then proceed immediately to the next step Incubate for 45 min at 37 C then for 5 min at 95 C then for at least 2 min at 4 C A Incubate the RNA hydrolysis reaction in a thermal cycler using the RNA Hydrolysis program that is shown in Table
32. el holder and add 1X TBE Buffer to the gel system and equilibrate to room temperature 1 Prepare NeutrAvidin and biotin labeled cDNA sample mix A B On ice prepare a NeutrAvidin solution of 2 mg mL in PBS For each sample to be tested prepare 2 aliquots of 1 uL fragmented and biotin labeled ss cDNA sample in a tube or well Heat the samples for 2 min at 70 C Centrifuge briefly to collect the reaction at the bottom of the tube or well then proceed immediately to the next step At room temperature add 5 uL of the 2 mg mL NeutrAvidin solution to one tube or well and add 5 uL of PBS to the other tube or well Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect contents of the tube and incubate for 5 min at room temperature 2 Separate the fragmented and labeled ss cDNA by size and stain A Prepare 10 bp and 100 bp DNA ladders by combining 1 uL of ladder and 7 uL of Nuclease free Water At room temperature add loading dye to all samples and DNA ladders to a final concentration of 1X loading dye Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect contents ofthe tube and proceed immediately to the next step Carefully load samples and ladders on gel Each well can hold a maximum of 20 uL Appendix A Gel Shift Assay 46 E Run the gel at 150 volts until the front dye almost reaches the bottom approximately 1 hr F Whilethe gel is running prepare
33. ent Setup hybridizations for one array strip at a time staggered by 1 5 hours so that washing and staining can occur immediately after completion of hybridization for each array strip the next day Recommended hybridization time is 20 1 hour 1 Navigate to the Hybridization tab on the GeneAtlas Software interface Chapter 3 WT Array Hybridization 38 AX Affymetrix HYBRIDIZATION Strip 2 Click the Strip button EJ The Add Strip Window appears Figure 3 14 Add Strip Scan or enter the bar code Bar Code Strip Name Time 30 Temperature 45 C 3 Scan or enter the Bar Code required of the array strip you registered The Strip Name field is automatically populated 4 With the hybridization tray and array strip already in the GeneAtlas Hybridization Station click Start in Figure 3 15 Add Strip Scan or enter the bar code Bar Code Strip Name Time 30 Temperature 45 C KOS Affymetrix HYBRIDIZATION Hyb001 00 00 27 i 48 C Chapter 3 WT Array Hybridization 39 yellow and the time begins to count up NOTE The software displays the hybridization time countdown This time is displayed with a white background Figure 3 16 When the countdown has completed the display turns XX Affymetrix HYBRIDIZATION Hyb001 i 48 C 5 When hybridization has completed click the Stop button
34. entially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as lab coat safety glasses and gloves Care should be taken to avoid contact with skin and eyes In case of contact with skin or eyes wash immediately with water See MSDS Material Safety Data Sheet for specific advice Chapter 1 WT PLUS Reagent Kit Assay Workflow 6 Figure 1 1 WT PLUS Amplification and Labeling Process Incubation Time 5 M AAAAAAAAAAAAAAA 3 First Strand cDNA Synthesis RT lt nnn TT 5 x 2hr i v 5 pr LAINIII 3 cM 2 4 Second Strand cDNA Synthesis DNA POLYMERASE amp RNASE H 1 hr 10 min 5 B3 3 TT CUM T7 RNA POLYMERASE cRNA Purification amp Quantitation N 5 NNN RT 8 esas ey E 2nd Cycle ss cDNA Synthesis v 2hr dUTP 5 NNN M Ses 3 3 _ Sd NT 5 Template RNA Removal v RNASE H 50 min dUTP 5 NNI eese re a 3 d Rr CT ss CDNA Purification amp Quantitation Fragmentation 1 hr Terminal Labeling 1 Ar Hybridization to WT Array NINN RNA NINN DNA RT Reverse Transcriptase NNN Random Primers DLR DNALabeling Reagent Biotin e 4 RNA Hydrolysis Kit Contents and Storage Chapter 1 WT PLUS Reagent Kit Table 1 1 GeneChip
35. g losses Table 2 6 First Strand Master Mix Component Volume for One Reaction First Strand Buffer 4 uL First Strand Enzyme 1 uL Total Volume 5 pL B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube Proceed immediately to the next step C On ice transfer 5 uL of the First Strand Master Mix to each tube or well 2 Add total RNA to each First Strand Master Mix aliquot A On ice add 5 uL of the total RNA Table 2 5 to each 5 uL tube or well containing the First Strand Master Mix for a final reaction volume of 10 uL See Prepare Total RNA Poly A RNA Control Mixture on page 14 for more information B Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the reaction at the bottom of the tube or well then proceed immediately to the next step 3 Incubate for 1 hr at 25 C then for 1 hr at 42 C then for at least 2 min at 4 C A Incubate the first strand synthesis reaction in a thermal cycler using the First Strand cDNA Synthesis program that is shown in Table 2 1 on page 11 B Immediately after the incubation centrifuge briefly to collect the first strand cDNA at the bottom of the tube or well Chapter2 Protocol 16 C Place the sample on ice for 2 min to cool the plastic then proceed immediately to Synthesize Second Strand cDNA on page 16 Lu IMPORTANT Transferring Second Strand Master Mix to hot plastics may signific
36. gram that is shown in Table 2 1 on page 11 C Atroom temperature prepare the Hybridization Master Mix in a nuclease free tube Combine the appropriate amount of components in the sequence shown in the table below Prepare the master mix for all the fragmented and biotin labeled ss cDNA samples in the experiment Include 10 overage to correct for pipetting losses Table 3 1 Hybridization Master Mix for a Single Reaction Component 49 or 64 100 or 81 4 169 Format Final Format Format Concentration Fragmented and Labeled ss DNA 5 2 ug 3 5 ug 2 3 ug 23 ng pL Control Oligo B2 3 nM 3 7 uL 2 5 uL 1 7 uL 50 pM 20X Hybridization Controls 11 uL 7 5 uL 5 uL 1 5 5 25 and 100 bioB bioC bioD cre pM respectively 2X Hybridization Mix 110 uL 75 uL 50 uL 1X DMSO 15 4 uL 10 5 uL 7 uL 7 Nuclease free Water 19 9 uL 13 5 uL 9 3 uL Total Volume 160 pL 109 pL 73 pL Please refer to specific probe array package insert for information on array format D Mix thoroughly by gently vortexing Centrifuge briefly to collect the mix and proceed immediately to the next step 2 Prepare Hybridization Cocktail A Atroom temperature add the appropriate amount of Hybridization Master Mix to each fragmented and biotin labeled ss cDNA sample to prepare Hybridization Cocktail Table 3 2 Hybridization Cocktail for a Single Array Component 49 or 64 Format 100 or 81 4 Format 169 Format Hybridization Master M
37. he sequence shown in the table below Prepare the master mix for all the fragmented and biotin labeled ss cDNA samples in the experiment f NOTE The 5X WT Hyb Add 1 solution is very viscous pipet slowly to ensure addition of the correct volume Mix well Vortex and centrifuge briefly 5 sec to collect liquid at the bottom of the tube Table 3 5 Hybridization Master Mix Component Volume for Volume for Final One Array Four Arrays Concentration Includes 10 Overage 5X WT Hyb Add 1 30 uL 132 uL 1X Control Oligonucleotide 1 5 uL 6 6 uL 30 pM B2 3 nM 20X Hybridization Controls 7 5 uL 33 uL 1 5 5 25 and 100 pM bioB bioC bioD cre respectively 15X WT Add 4 10 uL 44 uL 1X Total Volume 49 uL 215 6 pL C Mix thoroughly by gently vortexing Centrifuge briefly to collect the mix and proceed immediately to the next step 4 Prepare Hybridization Cocktail A At room temperature prepare the Hybridization Cocktail in the order as shown in Table 3 6 for all samples Table 3 6 Hybridization Cocktail for a Single Array Chapter 3 WT Array Hybridization 32 Component Volume for One Final Concentration Array Hybridization Master Mix 49 uL Fragmented and labeled ss cDNA 41 uL 23 ng uL 2 5X WT Hyb Add 6 60 uL 1X Total Volume 150 pL B If you are using a plate seal vortex and centrifuge briefly 5 sec to collect liquid at the bottom of the well If you are using t
38. ification Beads Mix the Purification Beads container by vortexing to resuspend the magnetic particles that may have settled Add 100 uL of the Purification Beads to each 60 uL cRNA sample mix by pipetting up and down and transfer to a well of a U bottom plate TIP a Any unused wells should be covered with a plate sealer so that the plate can safely be reused a Use multichannel pipette when processing multiple samples Mix well by pipetting up and down 10 times D Incubate for 10 min The cRNA in the sample binds to the Purification Beads during this incubation Move the plate to a magnetic stand to capture the Purification Beads When capture is complete after 5 min the mixture is transparent and the Purification Beads form pellets against the magnets in the magnetic stand The exact capture time depends on the magnetic stand that you use and the amount of cRNA generated by in vitro transcription Carefully aspirate and discard the supernatant without disturbing the Purification Beads Keep the plate on the magnetic stand 2 Wash the Purification Beads A While on the magnetic stand add 200 uL of 80 ethanol wash solution to each well and incubate for 30 sec Slowly aspirate and discard the 8096 ethanol wash solution without disturbing the Purification Beads Chapter2 Protocol 19 C Repeat Step A and Step B twice for a total of 3 washes with 200 uL of 80 ethanol wash solution Completely remove
39. ile Name column click in the box and enter a sample name and press Enter Enter a unique name for each of the four samples on the array strip 6 When complete click the Save and Proceed button EL The Save dialog box appears Figure 3 11 Figure 3 11 Save Dialog C APS Data Default Arr Folder Date Excel Protocol Template Workflow Create New Folder 05 26 2009 14 31 13 05 26 2009 14 31 13 05 26 2009 14 31 13 05 26 2009 14 31 13 05 26 2009 14 31 13 Chapter 3 WT Array Hybridization 37 7 In the Save dialog box click to select a folder in which to save your data Click OK Your files are saved to the selected folder and a confirmation message appears Figure 3 12 Figure 3 12 9 Sample Files Created as 8 Click OK to register additional array strips or click Go to Hybridization NOTE You may enter a total of four array strips during the registration process To add additional strips please repeat Step 3 through Step 8 9 Proceed to Hybridization Software Setup on page 37 Hybridization Software Setup All Affymetrix Array Strips to be processed must first be registered prior to setting up the hybridizations in the GeneAtlas Software Refer to Sample Registration on page 35 for instruction on registering array strips Lu IMPORTANT When hybridizing more than one array strip per day it is recommended to keep the hybridization time consist
40. ilibrate to room temperature before washing and staining NOTE Arrays can be stored in the Wash Buffer A at 4 C for up to 3 hr before proceeding with washing and staining Equilibrate arrays to room temperature before washing and staining Place vials into sample holders on the fluidics station A Place one amber vial containing 600 uL Stain Cocktail 1 in sample holder 1 B Place one clear vial containing 600 uL Stain Cocktail 2 in sample holder 2 C Place one clear vial containing 800 uL Array Holding Buffer in sample holder 3 Wash the arrays according to array type and components used for Hybridization Wash and Stain For HWS kits the protocols are Table 3 4 Fluidics Protocol 49 or 64 Format 100 or 81 4 Format 169 Format Fluidics Protocol FS450 0001 FS450 0002 FS450 0007 Check for air bubbles If there are air bubbles manually fill the array with Array Holding Buffer If there are no air bubbles cover both septa with 3 8 Tough Spots Inspect the array glass surface for dust and or other particulates and if necessary carefully wipe the surface with a clean lab wipe before scanning The instructions for using the scanner and scanning arrays can be found in the Affymetrix GeneChip Command Console User Manual P N 702569 Chapter 3 WT Array Hybridization 30 Array Strips Hybridization on the GeneAtlas Instrument This section outlines the basic steps involved in hybridizing array strip s on
41. ix 160 uL 109 uL 73 uL Fragmented and labeled ss cDNA 60 uL 5 2 ug 41 uL 3 5 ug 27 uL 2 3 ug Total Volume 220 pL 150 pL 100 pL This volume is 58 LL if a portion of the sample was set aside for gel shift analysis B Mix thoroughly by gently vortexing Centrifuge briefly to collect contents of the tube and proceed immediately to the next step C Incubate the hybridization cocktail reaction for 5 min at 99 C tubes or 95 C plates then for 5 min at 45 C in a thermal cycler using the Hybridization Cocktail program that is shown in Table 2 1 on page 11 Chapter 3 WT Array Hybridization 28 D After the incubation centrifuge briefly to collect contents of the tube and proceed immediately to the next step 3 Inject and hybridize array Figure 3 1 GeneChip Probe Array Plastic cartridge Front Probe array on glass substrate NOTE It is necessary to use two pipette tips when filling the probe array cartridge one for filling and the second to allow venting of air from the hybridization chamber A Insert a pipet tip into the upper right septum to allow for venting B Inject the appropriate amount see Table 3 3 of the specific sample into the array through one of the septa see Figure 3 1 for location of the septa on the array Table 3 3 Probe Array Cartridge Volumes for Hybridization Cocktail 49 or 64 Format 100 or 81 4 Format 169 Format Volume to Load on Array 200 uL
42. l Prepare Ovens Arrays and Sample Registration Files 1 Turn Affymetrix Hybridization Oven on set the temperature to 45 C and set the RPM to 60 Turn the rotation on and allow the oven to preheat 2 Equilibrate the arrays to room temperature immediately before use Label the array with the name of the sample that will be hybridized 3 Register the sample and array information into AGCC Target Hybridization Setup for Cartridge Arrays Reagents and Materials Required GeneChip Hybridization Wash and Stain Kit Not supplied For ordering information please refer to Table 1 4 on page 9 or the Affymetrix website u Pre Hybridization Mix n 2X Hybridization Mix o DMSO o Nuclease free Water o Stain Cocktail 1 o Stain Cocktail 2 n Array Holding Buffer u Wash Buffer A u Wash Buffer B GeneChip Hybridization Control Kit o 20X Eukaryotic Hybridization Controls bioB bioC bioD cre o Control Oligonucleotide B2 3 nM a Affymetrix WT Cartridge Array s Not supplied Chapter 3 WT Array Hybridization 27 Procedure 1 Prepare Hybridization Master Mix A At room temperature thaw the components listed in Table 3 1 x NOTE DMSO will solidify when stored at 2 8 C Ensure that the reagent is completely thawed before use We recommend to store DMSO at room temperature after the first use B Heat the 20X Hybridization Controls for 5 min at 65 C in a thermal cycler using the Hybridization Control pro
43. l Expected cRNA Size Distribution The expected cRNA profile is a distribution of sizes from 50 to 4500 nt with most of the cRNA sizes in the 200 to 2000 nt range The distribution is quite jagged and does not resemble the profile observed when using a traditional dT based amplification kit such as 3 IVT Express kit This step is optional Determine cRNA size distribution using a Bioanalyzer Affymetrix recommends analyzing cRNA size distribution using an Agilent 2100 Bioanalyzer a RNA 6000 Nano Kit PN5067 1511 and mRNA Nano Series II assay If there is sufficient yield then load approximately 300 ng of cRNA per well on the Bioanalyzer If there is insufficient yield then use as little as 200 ng of cRNA per well To analyze cRNA size using a Bioanalyzer follow the manufacturer s instructions EH TIP STOPPING POINT The purified cRNA samples can be stored overnight at 20 C Synthesize 2nd Cycle Single Stranded cDNA In this procedure sense strand cDNA is synthesized by the reverse transcription of cRNA using 2nd Cycle Primers The sense strand cDNA contains dUTP at a fixed ratio relative to dTTP 15 ug of cRNA is required for 2nd cycle single stranded cDNA synthesis 1 Prepare 15 ug of cRNA On ice prepare 625 ng uL cRNA This is equal to 15 ug cRNA in a volume of 24 uL If necessary use Nuclease free Water to bring the cRNA sample to 24 uL f NOTE High concentration cRNA samples gt 3000 ng L should be dilute
44. l Acad Sci USA 87 1663 1667 Revision History Description Section Update information for 30 Reaction Kit Kit Contents and Storage on page 7 Change volume of Nuclease free Water to 1 0 mL Kit Contents and Storage on page 7 Nuclease free Water for preparing 8096 ethanol Additional Reagents and Supplies Required Table 1 4 wash solution is added on page 9 Specify dilution requirement for high concentration Determine RNA Quantity on page 14 and Synthesize RNA samples First Strand cDNA on page 15 Specify long term storage for STOPPING POINT Chapter 2 Protocol Specify cRNA dilution requirement for concentration Assess cRNA Yield and Size Distribution on page 19 and measurement and reaction set up Synthesize 2nd Cycle Single Stranded cDNA on page 20
45. lock The clamp should open effortlessly Refer to Figure 3 6 Is IMPORTANT The hybridization temperature for WT GeneAtlas Array Strips is 48 C Figure 3 6 Opening the clamps on the GeneAtlas Hybridization Station 1 Push Down IMPORTANT The hybridization temperature for WT GeneAtlas Array Strips is 48 C f ll p 2 Lift Lever ull H Place the hybridization tray with the array strip into a clamp inside the Hybridization Station and close the clamp as shown in Figure 3 7 Chapter 3 WT Array Hybridization 35 Figure 3 7 Laterally inserting an array strip and closing the clamp of the GeneAtlas Hybridization Station IMPORTANT The hybridization temperature for WT GeneAtlas Array Strips is 48 C 6 Proceed to Hybridization Software Setup on page 37 GeneAtlas Software Setup Prior to setting up the target hybridization and processing the Affymetrix Array Strips on the GeneAtlas System each array strip must be registered and hybridizations setup in the GeneAtlas Software Sample Registration Sample registration enters array strip data into the GeneAtlas Software and saves and stores the Sample File on your computer The array strip barcode is scanned or entered and a Sample Name is input for each ofthe four samples on the array strip Additional information includes Probe Array Type and Probe Array position Hybridization Software Setup During the Hybridiza
46. nd cDNA Synthesis 42 C 105 C 25 C 60 min 42 C 60 min 4 C 2 min 10 uL Second Strand cDNA Synthesis RT or Lid open 16 C 60 min 65 C 10min 4 C 2 min 30 uL disable In Vitro Transcription cRNA 40 C 40 C oven 40 C 16 hr 4 C hold 60 uL Synthesis 2nd Cycle Primers cRNA 70 C 105 C 70 C 5 min 25 C 5 min 4 C 2 min 28 uL Annealing 2nd Cycle ss cDNA Synthesis 70 C 105 C 25 C 10 min 42 C 90 min 70 C 10 min 4 C hold 40 uL RNA Hydrolysis 70 C 105 C 37 C 45min 95 C 5 min 4 C hold 44 uL Fragmentation 93 C 105 C 37 C 60 min 93 C 2 min 4 C hold 48 uL Labeling 70 C 105 C 37 C 60min 70 C 10 min 4 C hold 60 uL Hybridization Control 65 C 105 C 65 C 5 min Variable Hybridization Cocktail 99 C 105 C 95 C or 45 C 5 min Variable 99 C 5 min For thermal cyclers that lack a programmable heated lid Reagent Preparation Handling kit components as follows Enzymes Mix by gently vortexing the tube followed by a brief centrifuge to collect contents of the tube then keep on ice Buffers and Primers Thaw on ice thoroughly vortex to dissolve precipitates followed by a brief centrifuge to collect contents of the tube If necessary warm the buffer s at lt 37 C for 1 to 2 min or until the precipitate is fully dissolved then keep on ice Purification Beads Allow to equilibrate at room temperature before use Prepare master mixes for each step of the procedure to save time improve re
47. oom temperature for 2 10 min before preparing the IVT Master Mix 1 Prepare IVT Master Mix NOTE This step is performed at room temperature A At room temperature prepare the IVT Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the second strand cDNA samples in the experiment Include 5 excess volume to correct for pipetting losses Table 2 8 IVT Master Mix Component Volume for One Reaction IVT Buffer 24 uL IVT Enzyme 6 uL Total Volume 30 pL D Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the mix at the bottom of the tube then proceed immediately to the next step At room temperature transfer 30 uL of the VT Master Mix to each 30 uL second strand cDNA sample for a final reaction volume of 60 uL Mix thoroughly by gently vortexing the tube Centrifuge briefly to collect the reaction at the bottom of the tube or well then proceed immediately to the next step 2 Incubate for 16 hr at 40 C then at 4 C A Incubate the IVT reaction in a thermal cycler using the In Vitro Transcription cRNA Synthesis program that is shown in Table 2 1 on page 11 B After the incubation centrifuge briefly to collect the cRNA at the bottom of the tube or well C Place the reaction on ice then proceed to Purify cRNA on page 18 or immediately freeze the samples at 20 C for s
48. producibility and minimize pipetting error Prepare Master Mixes as follows o Prepare only the amount needed for all samples in the experiment plus 5 overage to correct for pipetting losses when preparing the master mixes o Use non stick nuclease free tubes to prepare the master mix o Enzyme should be added last and just before adding the master mix to the reaction Return the components to the recommended storage temperature immediately after use Chapter2 Protocol 12 Prepare Control RNA Prepare Control RNA To verify that the reagents are working as expected a Control RNA sample 1 mg mL total RNA from HeLa cells is included with the kit To prepare the Control RNA for positive control reaction 1 On ice dispense 2 uL of the Control RNA in 78 uL of Nuclease free Water for a total volume of 80 uL 25 ng uL 2 Follow the Prepare Total RNA Poly A RNA Control Mixture on page 14 but use 2 uL of the diluted Control RNA 50 ng in the control reaction NOTE The positive control reaction should produce gt 15 ug of cRNA and gt 5 5 ug of 2nd cycle ss cDNA from 50 ng Control RNA Prepare Poly A RNA Controls NOTE a To include premixed controls from the GeneChip Poly A RNA Control Kit add the reagents to the total RNA samples Follow the Prepare Total RNA Poly A RNA Control Mixture on page 14 Affymetrix strongly recommends the use of Poly A RNA Controls for all reactions that will be hybridized to
49. r Expression Arrays Plates P N 702933 A IMPORTANT It is important not to bump the trays while loading them into the GeneTitan Instrument Droplets of the stain going onto the lid may result in a wicking action and the instrument gripper may be unable to remove the lids properly The remaining hybridization ready sample can be stored at 20 C after the Biorad Hardshell Plate using Aluminum Foil Gel Shift Assay The efficiency of the labeling procedure can be assessed using the following procedure This quality control protocol prevents hybridizing poorly labeled target onto the probe array The addition of biotin residues is monitored in a gel shift assay where the fragments are incubated with avidin prior to electrophoresis The nucleic acids are then detected by staining The procedure takes approximately 90 min to complete Table A 1 Additional Reagents Required Item Supplier P N XCell SureLock Mini Cell Life Technologies E10001 4 20 TBE Gel1 0 mm 12 well Life Technologies EC62252BOX Novex Hi Density TBE Sample Buffer 5X Life Technologies LC6678 TBE Buffer 5X Solution Affymetrix 75891 SYBR Gold Nucleic Acid Gel Stain Life Technologies 11494 10 bp DNA ladder and Life Technologies 10821 015 and 100 bp DNA ladder 15628 019 NeutrAvidin Protein Thermo Scientific 31000 PBS pH 7 2 Life Technologies 20012 027 Or equivalent lt NOTE Place a4 to 20 TBE gel into the g
50. rate at room temperature For each reaction 100 pL plus 10 overage will be needed a Prepare fresh dilutions of 80 ethanol wash solution each time from 100 ethanol Molecular Biology Grade or equivalent and Nuclease free Water in a nuclease free tube or container For each reaction 600 pL plus 10 overage will be needed a Transfer the cDNA sample to room temperature while preparing the Purification Beads NOTE a Occasionally the bead sample mixture may be brownish in color and not completely clear when placed on magnet In those situations switch to a different position of magnet on the magnetic stand a new magnetic stand or spin out pellets a This entire procedure is performed at room temperature ind ss cDNA to Purification Beads Mix the Purification Beads container by vortexing to resuspend the magnetic particles that may have settled Add 100 uL of Purification Beads to each 55 uL 2nd cycle ss cDNA sample mix by pipetting up and down and transfer to a well of a U bottom plate TIP a Any unused wells should be covered with a plate sealer so that the plate can safely be reused a Use multichannel pipette when processing multiple samples Add 150 uL of 100 ethanol to each 155 uL ss cDNA Beads sample Mix well by pipetting up and down 10 times Incubate for 20 min The ss cDNA in the sample binds to the Purification Beads during this incubation Move the plate to a magneti
51. ray Aliquot 105 uL of the Stain 1 into the GeneTitan Stain Tray 3 Use the anti static gun on the stain tray cover A Place a stain tray cover on the table top with the flat surface facing upward B Hold the Zerostat 3 anti static gun within 12 30 5 cm of the surface or object to be treated Squeeze the trigger slowly for about two seconds to emit a stream of positive ionized air over the surface of the object As the trigger is slowly released a negative flow of air ions is produced resulting in static neutralization C Repeat this procedure at several points across the surface covering the entire stain tray cover o Oon nau Pp 11 Chapter 3 WT Array Hybridization 44 After removing the static electricity place the cover on top Stain Tray 1 After repeating Step 1 aliquot 105 uL of the Stain 2 into the GeneTitan Stain Tray After repeating Step 3 place cover on top of Stain Tray 2 After repeating Step 1 aliquot 105 uL of the Stain 3 into the GeneTitan Stain Tray After repeating Step 3 place cover on top of Stain Tray 3 Aliquot 150 uL of the Array Holding Buffer into the GeneTitan Scan Tray identified with the label HT Scan Tray P N 500860 on the tray Use the fourth scan tray cover provided with the GeneTitan Consumable Upgrade kit to cover the Scan Tray Load all the consumables including the HT Array Plate into the GeneTitan Instrument as per instructions provided in the GeneTitan Instrument User Guide fo
52. se free Water at 20 C 4 C or room temp Do not freeze Tubes Organizer Plastic vinyl template for organization and storage of components in 9 x 9 array 81 places square wells 5 1 4 in x 5 1 4 in e g Nalgene CryoBox P N 5026 0909 or 7 Chapter 1 WT PLUS Reagent Kit 8 Required Materials Instruments Table 1 2 Instruments Required for Target Preparation Item Supplier Magnetic Stand 96 Agencourt SPRI Plate Super Magnet Plate Beckman Coulter Genomics P N A32782 Ambion Magnetic Stand 96 Life Technologies P N AM10027 96 well Magnetic Ring Stand Life Technologies P N AM10050 or equivalent magnetic stand Microcentrifuge Major Laboratory Supplier NanoDrop UV Vis Spectrophotometer Thermo Scientific or equivalent quantitation instrument Optional 2100 Bioanalyzer Agilent Technologies Inc or equivalent DNA and RNA sizing instrument Pipette Major Laboratory Supplier Thermal Cycler Various Vortex Mixer Major Laboratory Supplier 65 C heat block or oven for incubation of Major Laboratory Supplier Nuclease free Water during Purification Table 1 3 Instruments Required for Array Processing Instruments Supplier Part Number GeneChip System for Cartridge Arrays GeneChip Hybridization Oven 645 Affymetrix P N 00 0331 110 220V GeneChip Fluidics Station 450 Affymetrix P N 00 0079 GeneChip Scanner 3000 7G Affymetrix P N 00 0212 North America
53. sfer the supernatant which contains the eluted ss cDNA to a nuclease free tube mon Ww Place the purified ss cDNA samples on ice then proceed to Assess Single Stranded cDNA Yield and Size Distribution or immediately freeze the samples at 20 C for storage NOTE Minimal bead carryover will not inhibit subsequent enzymatic reactions EH TIP STOPPING POINT The purified ss cDNA samples can be stored overnight at 20 C For long term storage at 20 C we recommend not to proceed to the fragmentation and labeling reaction and store the samples as ss cDNA Assess Single Stranded cDNA Yield and Size Distribution Expected Single Stranded cDNA Yield During development of this kit using a wide variety of tissue types 15 ug of input cRNA yielded 5 5 to 15 ug of ss cDNA For most tissue types the recommended 15 ug of input cRNA should yield gt 5 5 ug of ss cDNA Determine Single Stranded DNA Yield by UV Absorbance Determine the concentration of a ss cDNA solution by measuring its absorbance at 260 nm Use Nuclease free Water as blank Affymetrix recommends using NanoDrop Spectrophotometers for convenience No dilutions or cuvettes are needed just use 1 5 uL of the cDNA sample directly Alternatively determine the ss cDNA concentration by diluting an aliquot of the preparation in Nuclease free Water and reading the absorbance in a traditional spectrophotometer at 260 nm Calculate the concentration in ug mL using the equa
54. the Hybridization Mix WT Hyb Add 2 3 and 5 are not used and are not part of the Hybridization Module 1 In preparation of the hybridization step prepare the following A Pull the array strip from storage at 4 C so that it can begin to equilibrate to room temperature B Gather one 1 hybridization tray per array strip C Set the temperature of the GeneAtlas Hybridization Station to 48 C Press the Start button Chapter 3 WT Array Hybridization 31 2 In preparation of the hybridization master mix prepare the following A Warm the following vials to room temperature on the bench 5X WT Hyb Add 1 15X WT Hyb Add 4 2 5X WT Hyb Add 6 B Vortex and centrifuge briefly 5 sec to collect contents of the tube C Remove the following tubes from the GeneChip Hybridization Control Kit and thaw at room temperature a Control Oligonucleotide B2 3 nM a 20X Eukaryotic Hybridization Controls Vortex and centrifuge briefly 5 sec to collect contents of the tube Keep the tubes of Control Oligonucleotide B2 3 nM and 20X Eukaryotic Hybridization Controls on ice 3 Prepare the Hybridization Master Mix amp Cocktail A Heat the 20X Hybridization Controls for 5 min at 65 C in a thermal cycler using the Hybridization Control program that is shown in Table 2 1 on page 11 At room temperature prepare the Hybridization Master Mix in a nuclease free tube Combine the appropriate amount of components in t
55. tion Software Setup the array strip to be processed is scanned and the GeneAtlas Hybridization Station is identified with hybridization time and temperature settings determined from installed library files For additional information please refer to GeneAtlas System User s Guide P N 08 0306 Sample Registration The following information provides general instructions for registering Affymetrix Array Strips in the GeneAtlas Software For detailed information on Sample Registration importing data from Excel and information on the wash stain and scan steps please refer to the GeneAtlas System User s Guide P N 08 0246 1 Click Start Programs Affymetrix GeneAtlas to launch the GeneAtlas Software Chapter 3 WT Array Hybridization 36 2 Click the Registration tab Figure 3 8 appears eaa Affymetrix REGISTRATION Import Template MP Tm No Strips Added 3 Click the Strip button ES The Add Strip Window appears Figure 3 9 Add Strip Bar Code Strip Name 4 Enter or scan the array strip Bar Code and enter a Strip Name then click Add The array strip is added and appears in the Registration window Figure 3 10 KOS Affymetrix REGISTRATION Utility Actions v Import Template a Save and Proceed gt gt ES Sample File Name Probe Array Type Probe Array Position text text text 18 al Testi 3 5500251834075263864806 18 a3 18 a4 5 Under the Sample F
56. tion below 1 A 33 ug DNA mL Aso X dilution factor x 33 ug DNA mL NOTE The equation above applies only to single stranded cDNA Optional Expected Single Stranded cDNA Size Distribution The expected cDNA profile does not resemble the cRNA profile The median cDNA size is approximately 400 nt This step is optional Chapter2 Protocol 24 Determine Single Stranded cDNA Size Distribution Using a Bioanalyzer Affymetrix recommends analyzing cDNA size distribution using an Agilent 2100 Bioanalyzer a RNA 6000 Nano Kit PN5067 1511 and mRNA Nano Series II assay If there is sufficient yield load approximately 250 ng of cDNA per well If there is insufficient yield then use as little as 200 ng of cDNA per well To analyze cDNA size using a bioanalyzer follow the manufacturer s instructions EH TIP STOPPING POINT The purified ss cDNA samples can be stored overnight at 20 C For long term storage at 20 C we recommend not to proceed to the fragmentation and labeling reaction and store the samples as ss cDNA Fragment and Label Single Stranded cDNA In this procedure the purified sense strand cDNA is fragmented by uracil DNA glycosylase UDG and apurinic apyrimidinic endonuclease 1 APE 1 at the unnatural dUTP residues and breaks the DNA strand The fragmented cDNA is labeled by terminal deoxynucleotidyl transferase TdT using the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin
57. to calculate RIN go to www genomics agilent com With denaturing agarose gel electrophoresis and nucleic acid staining you separate and make visible the 28S and 18S rRNA bands The mRNA is likely to be full length if the 28S and 18S rRNA bands are resolved into two discrete bands that have no significant smearing below each band 28S rRNA band intensity is approximately twice that of the 18S rRNA band Determine RNA Quantity Consider both the type and amount of sample RNA that are available when planning your experiment Because mRNA content varies significantly with tissue type determine the total RNA input empirically for each tissue type or experimental condition The recommended total RNA inputs in Table 2 4 are based on total RNA from HeLa cells Use these values as reference points for determining your optimal RNA input x NOTE Avoid pipetting solutions less than 2 pL in volume to maintain precision and consistency High concentration RNA samples should be pre diluted with Nuclease free Water before adding to first strand cDNA synthesis reaction Table 2 4 Input RNA Limits RNA Input Total RNA Recommended 100 ng Minimum 50 ng Maximum 500 ng Prepare Total RNA Poly A RNA Control Mixture Prepare total RNA according to your laboratory s procedure A maximum of 5 uL total RNA can be added to first strand synthesis reaction If you are adding Poly A Spike Controls to your RNA the volume of RNA must be
58. torage EH TIP STOPPING POINT The cRNA samples can be stored overnight at 20 C Purify cRNA In thi Chapter 2 Protocol 18 S procedure enzymes salts inorganic phosphates and unincorporated nucleotides are removed to prepare the cRNA for 2nd cycle single stranded cDNA synthesis Beginning the c es 1 B A RNA Purification IMPORTANT a Preheat the Nuclease free Water in a heat block or thermal cycler to 65 C for at least 10 min Mix the Purification Beads thoroughly by vortexing before use to ensure that they are fully dispersed Transfer the appropriate amount of Purification Beads to a nuclease free tube or container and allow the Purification Beads to equilibrate at room temperature For each reaction 100 pL plus 10 overage will be needed a Prepare fresh dilutions of 80 ethanol wash solution each time from 100 ethanol Molecular Biology Grade or equivalent and Nuclease free Water in a nuclease free tube or container For each reaction 600 pL plus 10 overage will be needed a Transfer the cRNA sample to room temperature while preparing the Purification Beads NOTE a Occasionally the bead sample mixture may be brownish in color and not completely clear when placed on magnet In those situations switch to a different position of magnet on the magnetic stand a new magnetic stand or spin out pellets a This entire procedure is performed at room temperature ind cRNA to Pur
59. tray Figure 3 4 Placing the array strip into the hybridization tray Alignment Marks Thick D Optional the remainder of the hybridization cocktail Master Mix can be stored at 20 C to supplement Hybridization Cocktail volume should a rehybridization be necessary CAUTION Be very careful not to scratch damage the array surface Chapter 3 WT Array Hybridization 34 E TIP To avoid any possible mix ups the hybridization tray and array strip should be labeled on the white label if more than 1 array strip is processed overnight E Bring the hybridization tray to just above eye level and look at the underside of the hybridization tray to check for bubbles CAUTION Be careful not to tip the hybridization tray to avoid spilling Lu IMPORTANT Insertion of the array strip and air bubble removal should be performed quickly to avoid drying of the array surface F If an air bubble is observed separate the array strip from the hybridization tray and remove air bubbles Place array strip back into hybridization tray and recheck for air bubbles G Open a Hybridization Station clamp by applying pressure to the top of the clamp while gently squeezing inward While squeezing lift the clamp to open Figure 3 6 n WARNING Do not force the GeneAtlas Hybridization clamps up To open press down on the top of the clamp and simultaneously slightly lift the protruding lever to un
60. ttle Amount Required for Fluid Type One 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 3 10 Volumes Required to Process WT Array Plates per Run Number of Plates that can be Processed using the GeneTitan Hybridization Wash and Stain s Kit for WT Array Plates P N 901622 Amount Required for Reagent One Array Plate 16 Format 24 Format 96 Format Wash A 920 mL 1 1 1 Wash B 300 mL 1 1 1 Stain 1 and 3 105 uL well 6 4 1 Stain 2 105 uL well 6 4 1 Array Holding Buffer 150 uL well 6 4 1 IMPORTANT The instrument must have a minimum of 450 mL of Wash B in the Wash B reservoir of the instrument for each WT Array Plate prior to starting Hyb Wash Stain and Scan process The waste bottle should be empty Processing WT Array Plates on the GeneTitan Instrument 1 Use the anti static gun on the wells of the stain tray labeled GeneTitan Stain Tray P N 501025 A Place a stain tray on the table top B Hold the Zerostat 3 anti static gun within 12 30 5 cm of the surface or object to be treated Squeeze the trigger slowly for about two seconds to emit a stream of positive ionized air over the surface of the object As the trigger is slowly released a negative flow of air ions is produced resulting in static neutralization C Repeat this procedure at several points across the surface of the stain t
61. ubes vortex and centrifuge briefly 5 sec to collect contents of the tube C Incubate the hybridization cocktail reaction for 5 min at 99 C tubes or 95 C plates then for 5 min at 45 C in a thermal cycler using the Hybridization Cocktail program that is shown in Table 2 1 on page 11 D After the incubation centrifuge briefly to collect contents of the tube or well and proceed immediately to the next step 5 Array Strip Sample Hybridization A Apply 120 uL of hybridization cocktail to the middle of the appropriate wells of a new clean hybridization tray see Figure 3 2 Lu IMPORTANT Do not add more than 120 uL of hybridization cocktail to the wells as that could result in cross contamination of the samples Figure 3 2 Location of the sample wells on the hybridization tray Add sample to these four wells only A C E G DO NOT add sample to the space in the center of the hybridization tray B Carefully remove the array strip and protective cover from its foil pouch and place on bench Figure 3 3 Lu IMPORTANT Leave array strip in protective cover Chapter 3 WT Array Hybridization 33 Figure 3 3 Array Strip in protective tray HG U219 997 10088 C Place the array strip into the hybridization tray containing the hybridization cocktail samples Figure 3 4 Refer to Figure 3 5 for proper orientation of the array strip in the hybridization

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