User Manual, GeneChip® Whole Transcript WT
Contents
1. Primers Poly A RNA Controls 33 Procedure C Preparation of total RNA T7 N Primers Poly A RNA Controls stat gz sa SQ AA Rac n n D bans be Wa ahd bin d KA wks 34 Hybridizati N scese tee Tr rr 35 FAO PC nn 37 WT Sense Target Labeling Assay kk kK KK KK KK R isle 37 Array Hybridization Washing Staining and Scanning 40 Gel Shitt ASSAY xix posce eee w kK KK KK KK KK KK KK KK KK KK es 43 Fluidics Protocols And Fluidics Scripts for GeneChip ST ArrayS kk kk kk kk kk kK KK KK KK KK KK KK k 45 Fluidics Protocols lt 3 4 lt 445a oll bul RR RR Rs 45 Fluidics ScHpts si odia ddr eee 540534455405 a a nd de n bed 45 Overview The Affymetrix GeneChip Whole Transcript WT Sense Target Labeling Assay is designed to generate amplified and biotinylated sense strand DNA targets from the entire expressed genome without bias This assay and associated reagents have been optimized specifically for use with the GeneChip ST Arrays where ST stands for Sense Target and the probes on the arrays have been selected to be distributed throughout the entire length of each transcript x NOTE The WT Assay is not compatible with GeneChip arrays designed to focus on the 3 ends of the transcripts For the 3 arrays continue to follow the protocols detailed in the GeneChip Expression Analysis Technical Manual P N 900223 This manual describes in detail two con2. Amplification Kit Sample Cleanup Module WT Terminal Labeling 1 Kit Hybridization Control Kit Hybridization Wash and 4 Stain Kit Reagent Kits Poly A RNA Figure 1 1 GeneChip Whole Transcript Sense Target Labeling Assay Total RNA Sample STTTTTTTTTTT aaa 3 n d Poly A RNA Controls Control addition rRNA reduction RNA concentration T7 N e Primers M 9 NNN mm 5 STITITTITTITTT2a a I I J Il i I N N N azm si 1st cycle 15t strand cDNA synthesis 1st cycle 24 strand cDNA synthesis 5 TTITTTTTTTTT m n 3 SO0TD000T0000I 5 Un labeled Ribonucleotides 3 LNL LLILILILILIL_I_ILI 5 1st cycle in vitro transcription 15t cycle cleanup of antisense RNA cRNA Random Primers dUTP 5 U 3 3LLLLLLL LL EG 5 cRNA hydrolysis P d RNase H 2nd cycle 1st strand cDNA synthesis Cleanup of sense strand DNA r UDG Fragmentation xs APE 1 l TU i i TdT Terminal labeling DNA Labeling Reagent e e T li Il II od Hybridization Controls 4 Hybridization Stain Cocktail 1 Stain Cocktail 2 Washing Staining Scanning Legend 11111 RNA IT IT DNA NENN T7 promoter Approximate ExperimentTime 2 5 hours Overnight 0 5 hours 1 5 hours 1 hour 0 5 hours 1 5 hours 1 5 hours 16 hours 1 5 hours 10 35 minutes Biotin The rRNA reduction step is omitted in the 100 ng Total RNA Labeling P
3. Beads that are coated with streptavidin The unbound fraction represents the RNA with rRNA species reduced The sample is then concentrated before target labeling using the IVT cRNA Cleanup Kit Consult the handbook included in the RiboMinus Kit from Invitrogen for more details 2 Why is Betaine added to the RiboMinus Hybridization Buffer Betaine increases the hybridization stringency It equalizes the GC T and AT T so the background non specific non rRNA hybridization to the RiboMinus probes due to high GC content may be reduced 3 Why do you choose to use an rRNA reduction strategy but not a poly A mRNA specific selection protocol It has been shown that a portion of the transcripts in total RNA do not necessarily contain Poly A tails therefore they will be excluded by a poly A RNA positive selection technique The rRNA reduction approach will also make the protocol more robust in handling smaller amounts of total RNA samples as little as 1 ug 4 What is the recommendation on how the total RNA samples should be prepared for this assay A standard preparation method should be used as recommended for the current GeneChip Human Genome U133 Arrays The quality assessment metrics including the Bioanalyzer trace and O D ratios should remain unchanged 38 GeneChip Whole Transcript Sense Target Labeling Assay Manual 5 Does genomic DNA contamination in the sample interfere with the results and how do I monitor the degree
4. HG U133 arrays The WT Sense Target Labeling Assay is optimized to produce targets specifically for hybridization to ST array type of design The target is in the sense orientation and the GeneChip Human Genome U133 Plus 2 0 Array is designed to be compatible with anti sense targets Therefore it is not recommended to mix and match the assays and the array types Can I use this protocol for prokaryotic arrays This has not been tested at the moment therefore it is not recommended to use the protocol for any application other than on ST arrays How does this protocol perform on partially degraded samples Utilizing the WT assay for partially degraded samples may be an attractive strategy for profiling these samples However it has not been tested thus far in development therefore it is recommended that only high quality total RNA samples should be used Array Hybridization Washing Staining and Scanning 18 19 20 Why is there no pre hybridization step for the arrays using the targets from the WT Assay The pre hybridization step was required for the 3 target in the GeneChip Expression Analysis Technical Manual No pre hybridization step is necessary for the WT targets There are many differences between the WT targets and the 3 targets in terms of the nature of the molecules DNA vs RNA as well as labeling molecule and hybridization cocktail makeup It has been found that pre hybridization is not necessary fo
5. RNase free tube and leave on ice Wash the beads by re suspending them in 50 uL 50 uL of Hybridization Buffer with Betaine and incubate at 50 C for 5 minutes Place the tube in the magnetic stand for 1 to 2 minutes transfer the supernatant and combine with the supernatant in the tube from Procedure E Step 4 The total volume of the rRNA reduced sample is approximately 100 uL 150 uL Chapter 2 rRNA Reduction and Preparation of Total RNA with Diluted Poly A RNA Controls 19 Procedure F Concentration This Procedure requires the use of the GeneChip IVT cRNA Cleanup Kit NOTE This Procedure uses the IVT cRNA Cleanup Spin Columns in place of the 10 11 purification columns as part of the RiboMinus Kit Carefully follow the protocol described here for best results Proceed to the cleanup procedure using the IVT cRNA Cleanup Spin Columns from the IVT cRNA Cleanup Kit following the protocol described below Use the volumes between parenthesis if the starting amount of total RNA is 2 pg If not already done add 20 mL of Ethanol 100 to the cRNA Wash Buffer supplied in the IVT cRNA Cleanup Kit Add 350 uL 525 uL of cRNA Binding Buffer to each rRNA reduced sample from Procedure E and vortex for 3 seconds Add 250 uL 375 uL of 100 ethanol to each reaction and flick the tube to mix Apply the sample to the IVT cRNA Cleanup Spin Column sitting in a 2 mL Collection Tube IMPORTANT If the starting amount of
6. Technology 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 Use of the GeneChip WT cDNA Synthesis and Amplification Kit in accordance with the instructions provided is accompanied by a limited license to U S Patent Nos 5 716 785 5 891 636 6 291 170 and 5 545 522 Users who do not purchase this Kit may be required to obtain a license under these patents or to purchase another licensed kit Reagent products may also be covered by one or more of the following patents U S Patent Nos 6 864 059 Copyright 2005 2009 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Overview Lua uem aeuo d hance CUR MON kk kk kk kk aod kk ata lk kk 1 Whole Transcript Sense Target Labeling Assay Schematic 3 Materials ansas sa E eui e TUTO tE mala a RUE 4 Necessary REAGENTS ouai aan E A ala a a a A ag See 4 Miscellaneous Reagents kk kk kk kk kk KK KK KK KK KK KK KK KI KK KI KK KK 7 Miscellaneous Supplies kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK 8 Instr iTientS serm kanal n do RE eee Yon qur Pee hl nd n ee E d 9 Suggested Workflow kk kk kk kk kk kK KK KK KK KK KK KK KI KK KK RIK KK KO 10 rRNA Reduction and Preparation of Total RNA with Diluted Poly A RNA Controls 60 08 eedem E kk kK KK KK KK eae e
7. beads and wash solutions proportionately and carrying out the wash steps in a single tube Make sure to gently flick the tube with the beads several times for a thorough mixing after each step Use the magnetic stand as described under Step 4 if the beads remain attached to the wall of the tube Following the 3rd Wash Procedure D Step 6 the resuspended beads can then be aliquoted to individual tubes as described in Procedure D Step 7 before proceeding to rRNA Reduction for individual samples 18 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure E rRNA Reduction This Procedure requires the use of the RiboMinus Human Mouse Transcriptome Isolation Kit that needs to be obtained directly from Invitrogen Two heating blocks are required one at 37 C and the other at 50 C Use the volumes between parenthesis if the starting amount of total RNA is 2 pg 1 Transfer the ice cooled hybridized sample prepared in Procedure C to the beads prepared in Procedure D mix well and briefly spin Incubate the tube with the mixture at 37 C for 10 minutes in a heating block After 5 minutes of incubation gently flick mix the tube Briefly spin and place the tube in the magnetic stand for 1 to 2 minutes to obtain the rRNA probe pellet x NOTE The supernatant contains the rRNA Reduced Total RNA Poly A RNA Controls Mix 4 With the tube in the magnetic stand transfer the supernatant to a 1 5 mL non stick
8. of its effect By titrating genomic DNA back into the total RNA samples and monitoring the deterioration of the array data it was determined during development of the assay that a moderate amount of genomic DNA contamination will only have minimum effect on the array results Therefore routine RNA isolation techniques coupled with DNase treatment should yield sufficiently high quality sample for analysis on the GeneChip ST arrays 6 What starting material is needed for the assay One ug of total RNA per sample is the recommended starting quantity if following the standard protocol with an up front rRNA removal procedure Less total RNA 100 to 300 ng can be used with an alternative protocol without the rRNA reduction process however it is anticipated that the array detection sensitivity and specificity will be compromised at the exon level Follow recommendations on the optimal assay for each array as described in Chapter 1 7 Canluse more than 1 yg or 100 ng of total RNA While 1 ug of total RNA is the recommended amount when employing the 1 ug Total RNA Labeling Protocol a range of 1 to 2 ug can be used Higher amount of input total RNA typically result in modest increases in cRNA yield However when using 2 ug of total RNA it 1s highly recommended to scale up the RiboMinus reagents appropriately when carrying out the rRNA reduction step When employing the 100 ng Total RNA Labeling Protocol 100 to 300 ng of total RNA may be used
9. the use of the GeneChip Poly A RNA Control Kit The quality of the starting RNA sample is essential to the overall success of the analysis Since the most appropriate protocol for the isolation of RNA can be source dependent we recommend using a protocol that has been established for the tissues or cells being used In the absence of an established protocol using one of the commercially available kits designed for RNA isolation is suggested m IMPORTANT When using a commercial kit follow the manufacturer s instructions for RNA isolation The following protocol requires a minimum of 1 ug of total RNA as starting material and the concentration should not fall below 0 31 ug uL In other words the 1 ug of total RNA should be suspended in a maximum of 3 2 uL of solution in volume One to 2 ug of total RNA may be used but the RNA concentration must be high enough because the maximum volume of total RNA is 3 2 uL 1 The Poly A RNA controls are provided as a concentrated stock of 4 different transcripts at staggered concentrations Dilution buffer is supplied with the kit to prepare the appropriate dilutions based on Table 2 1 Use non stick RNase free microfuge tubes for all dilutions Keep the tubes on ice at all times Table 2 1 Poly A RNA Control Stock Serial Dilutions Starting Amount of Total RNA Serial Dilutions Volume into Sample First Second Third 1 ug 1 20 1 50 1 50 2 uL 2 ug 1 20 1 50 1 25 2 pL 12 Ge
10. total RNA is 2 yg the total supernatant volume will exceed the capacity of the column Apply 700 uL centrifuge for 15 seconds at gt 8 000 x g Discard the flow through Place the column back in to 2 mL collection tube and apply the remaining sample 350 pL Continue to Step 6 Centrifuge for 15 seconds at 2 8 000 x g Discard the flow through Transfer the IVT cRNA Cleanup Spin Column to a new 2 mL Collection Tube Add 500 uL of cRNA Wash Buffer and centrifuge for 15 seconds at gt 8 000 x g Discard the flow through Wash again with 500 uL of 80 v v ethanol Centrifuge for 15 seconds at 2 8 000 x g and discard the flow through Open column cap and spin at lt 25 000 x g maximum speed for 5 minutes with the cap left open Transfer the IVT cRNA Cleanup Spin Column to a new 1 5 mL Collection Tube and add 11 uL of RNase free Water directly to the membrane Spin at 25 000 x g maximum speed for 1 minute The eluted rRNA Reduced Total RNA Poly A RNA Controls Mix is 9 8 uL Keep sample on ice Proceed with Procedure G Analysis with Bioanalyzer and Chapter 3 Procedure A 1 NOTE Store eluted rRNA reduced total RNA at 80 C if not proceeding immediately to Procedure G Analysis with Bioanalyzer and Chapter 3 Procedure A 20 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure G Analysis with Bioanalyzer 1 Use 1 uL of the concentrated sample to check its quality by running the Eu
11. AX v Affymetrix User Manual GeneChip Whole Transcript WT Sense Target Labeling Assay P N 701880 Rev 5 For research use only Not for use in diagnostic procedures Trademarks s Affymetrix GeneChip AY HuSNP GenFlex Flying Objective CustomExpress CustomSeq NetAffx Tools To Take You As Far As Your Vision The Way Ahead Powered by Affymetrix GeneChip compatible Command Console Genotyping Console and GeneTitan are 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 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 and or sold under license from Oxford Gene
12. NA Cleanup Kit Affymetrix 900547 30 Rxn Contains a IVT cRNA Cleanup Spin Columns a IVT cRNA Binding Buffer n IVT cRNA Wash Buffer 5 mL concentrate a RNase free Water o 1 5 mL Collection Tubes for elution a 2 mL Collection Tubes GeneChip Sample Cleanup Module Affymetrix 900371 30 Rxn Contains a cDNA Cleanup Spin Columns a cDNA Binding Buffer n cDNA Wash Buffer 6mL concentrate o cDNA Elution Buffer a IVT cRNA Cleanup Spin Columns a IVT cRNA Binding Buffer n IVT cRNA Wash Buffer 5 mL concentrate a RNase free Water n 1 5 mL Collection Tubes for elution a 2 mL Collection Tubes n 5X Fragmentation Buffer Hybridization Stain and Wash GeneChip Hybridization Control Kit Affymetrix 900454 30 Rxn or Contains 900457 150 Rxn o 20X Hybridization Controls a 3 nM Control Oligo B2 GeneChip Hybridization Wash and Stain Kit 30 reactions Affymetrix 900720 30 Rxn Miscellaneous Reagents Table 1 3 Miscellaneous Reagents Chapter 1 Overview 7 Materials Source P N Miscellaneous Reagents Absolute ethanol Gold Shield N A Chemical Co RNA 6000 Nano Kit Agilent 5067 1511 Gel Shift Assay Optional Novex XCell SureLock Mini Cell Invitrogen EI0001 TBE Gel 4 2096 1 0 mm 12 well Invitrogen EC62252 Novex Hi Density TBE Sample Buffer 5X Invitrogen LC6678 10X TBE Buffer Cambrex 50843 SYBR Gold Invitrogen S 11494 10 bp DNA ladder and Invitrogen 10821 015 100 bp DNA ladder 15628 019 Im
13. NA Fragmentation Buffer 4 8 uL UDG 10 U uL 1 0 uL APE 1 1 000 U uL 1 0 uL Total Volume 16 8 uL 3 Add 16 8 uL of the above Fragmentation Master Mix to the samples prepared in Step 1 Flick or gently vortex the tubes and spin down 4 Incubate the reactions at 37 C for 60 minutes 93 C for 2 minutes 4 C for at least 2 minutes 5 Flick mix spin down the tubes and transfer 45 uL of the sample to a new 0 2 mL strip tube The remainder of the 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 See Figure 3 1 as an example of typical results on fragmented samples 6 Ifthe samples are not labeled immediately store the fragmented Single Stranded DNA at 20 C 30 GeneChip Whole Transcript Sense Target Labeling Assay Manual Fluorescence 14 19 24 29 34 33 tt 49 54 Time seconds Chapter 3 1 ug Total RNA Target Labeling Protocol 31 Procedure H Labeling of Fragmented Single Stranded DNA This Procedure requires the use of the GeneChip WT Terminal Labeling Kit 1 Prepare the labeling reactions as listed in Table 3 9 A master mix using the 5X TdT Buffer TdT and DNA Labeling reagent can be prepared just before aliquoting 15 uL into the 0 2 mL strip tubes containing the 45 uL of Fragm
14. Primers 2 5 ug uL 2uL 2 uL Diluted Poly A RNA Controls 3rd dilution 1 50 2 uL 6 uL RNase free Water 16 uL 12 uL Total Volume 20 uL 20 uL 2 Flick mix the solution spin down and place on ice 34 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure C Preparation of total RNA T7 N Primers Poly A RNA Controls 1 Mix total RNA and the T7 N Primers Poly A RNA Controls solution as listed in Table 4 2 Table 4 2 First Cycle Total RNA Primer Poly A RNA Controls Component Volume in 1 Rxn Total RNA 100 300 ng variable T7 N Primers Poly A RNA Controls Solution 2 uL RNase free Water up to 5 uL Total Volume 5 uL 2 Flick mix spin down the tube and incubate for 5 minutes at 70 C Then cool the sample for at least 2 minutes at 4 C and spin down 3 Place on ice for use in the section below Follow the same protocol as described in Chapter 3 Procedures B H starting on page 22 for the remainder of the Whole Transcript Sense Target Labeling Assay Hybridization This Procedure requires the use of the GeneChip Hybridization Wash and Stain Kit Three heating blocks are required one at 65 C one at 99 C and the third one at 45 C 1 Prepare the Hybridization Cocktail in a 1 5 mL RNase free microfuge tube as shown in Table 5 1 Table 5 1 Hybridization Cocktail Component Volume for One Volume for One Final 49 64 Format Array 169 Format Array Concentrat
15. This single stranded DNA sample is then treated with a combination of uracil DNA glycosylase UDG and apurinic apyrimidinic endonuclease 1 APE 1 that specifically recognizes the unnatural dUTP residues and breaks the DNA strand DNA is labeled by terminal deoxynucleotidyl transferase TdT with the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin Following the recommended procedures sufficient target is anticipated to be generated for hybridization to a single array Follow the instructions closely for the most optimal results As an Affymetrix GeneChip microarray user your feedback is welcome Please contact your technical support representative with any input on how we can improve this resource Table 1 1 Two Configurations of the WT Sense Target Labeling Assay 1 ug Total RNA Labeling Protocol 100 ng Total RNA Labeling Protocol Recommended Amount of Starting 1 ug Total RNA 100 ng Total RNA Material Acceptable Range of Input Amount 1 2 ug 100 300 ng Procedural Difference Requires rRNA Reduction with Omits rRNA Reduction RiboMinus Kit Exon ST Arrays Recommended Not Optimal Gene ST Arrays Acceptable Recommended Chapter 1 Overview Whole Transcript Sense Target Labeling Assay Schematic 3 Affymetrix wW Poly A RNA Control Kit IVT cRNA Cleanup Kit WT cDNA Synthesisand Amplification Kit Sample Cleanup 4 Module WT cDNA Synthesis and
16. Total RNA Labeling Protocol is acceptable for use with the GeneChip Gene 1 0 ST Arrays When omitting the RiboMinus procedure using 100 ng of total RNA as input has been shown to generate sufficient cRNA from a diverse set of RNA sources However for some RNAs that yield less cRNA increasing the amount of total RNA used can result in a modest increase in cRNA made Target generated from a range of 100 to 300 ng of input total RNA has demonstrated equivalent array performance 2 GeneChip Whole Transcript Sense Target Labeling Assay Manual For more information regarding the performances of the two protocols on Gene 1 0 ST Arrays refer to the Whole Transcript Sense Target Labeling Assay Performance white paper As outlined in Figure 1 1 following the rRNA reduction procedure the two protocols merge where double stranded cDNA is synthesized with random hexamers tagged with a T7 promoter sequence The double stranded cDNA is subsequently used as a template and amplified by T7 RNA polymerase producing many copies of antisense cRNA In the second cycle of cDNA synthesis random hexamers are used to prime reverse transcription of the cRNA from the first cycle to produce single stranded DNA in the sense orientation In order to reproducibly fragment the single stranded DNA and improve the robustness of the assay a novel approach is utilized where dUTP is incorporated in the DNA during the second cycle first strand reverse transcription reaction
17. as input 8 Whatis the typical cRNA yield after the IVT reaction in the first cycle Starting with 1 ug of total RNA following the standard protocol gt 8 ug of cRNA is routinely obtained cRNA yields will depend mainly on the cell line or tissue used Starting with 100 ng oftotal RNA without the RiboMinus rRNA removal step typically 15 to 40 ug of cRNA can be generated after cRNA cleanup 9 What is the basic principle of the single stranded DNA fragmentation and labeling procedure Using cRNA generated from the IVT reaction at the end of the first cycle of the assay as a template single stranded DNA is synthesized using random primers and the dUTP dNTP mix The resulting single stranded DNA ss DNA containing the unnatural uracil base is then treated with Uracil DNA Glycosylase which specifically removes the uracil residue from the ss DNA molecules In the same reaction the APE 1 enzyme then cleaves the phosphodiester backbone where the base is missing leaving a 3 hydroxyl and a 5 deoxyribose phosphate terminus 10 11 12 13 14 Appendix A FAQ 39 What is the basic component in the DNA Labeling Reagent The key labeling molecule in the DNA Labeling Reagent is Biotin Allonamide Triphosphate See the structure below O O H O HN O N Li OsP O Y 7 7v N Duel NH O S HO What is the expected length of the fragmented DNA target On a Bioanalyzer the fragmented single stranded DNA target
18. ately proceeding to the next Procedure First Cycle cRNA Synthesis and Cleanup NOTE Keeping the reaction at 4 C longer than 10 minutes may result in reduced cRNA yields 24 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure D First Cycle cRNA Synthesis and Cleanup This Procedure requires the use of the GeneChip WT cDNA Amplification Kit and the GeneChip Sample Cleanup Module 1 Ina separate tube assemble the IVT Master Mix at room temperature as listed in Table 3 4 Add the IVT Enzyme Mix to the master mix last and proceed immediately to aliquot into the tubes from Procedure C Step 5 as described in Step 5 below NOTE If a white precipitate is still present in the 10X IVT Buffer after thawing incubate the tube at 37 C until the precipitate gets dissolved Do not assemble the reaction on ice since the spermidine in the 10X IVT Buffer can lead to precipitation of the template DNA Table 3 4 First Cycle IVT Master Mix Component Volume in 1 Rxn 10X IVT Buffer 5 0 uL IVT NTP Mix 20 0 uL IVT Enzyme Mix 5 0 uL Total Volume 30 0 uL 2 Transfer 30 uL of the IVT Master Mix to each First Cycle cDNA Synthesis Reaction sample from Procedure C to a final volume of 50 uL Flick mix the solution and briefly spin in a microfuge 3 Incubate the reaction for 16 hours at 37 C To prevent condensation that may result from water bath style incubators incubations are best perf
19. b Wash 2 6 cycles of 15 mixes cycle with Wash Buffer B at 50 C Stain Stain the probe array for 5 minutes in SAPE solution at 35 C Post Stain Wash 10 cycles of 4 mixes cycle with Wash Buffer A at 30 C 2nd Stain Stain the probe array for 5 minutes in antibody solution at 35 C 3rd Stain Stain the probe array for 5 minutes in SAPE solution at 35 C Final Wash 15 cycles of 4 mixes cycle with Wash Buffer A at 35 C Holding Buffer Fill the probe array with Array Holding Buffer Wash Buffer A non stringent wash buffer a Wash Buffer B stringent wash buffer Fluidics Scripts Table C 2 Fluidics Scripts for GeneChip ST Array Types Array Format Fluidics Script Protocol 49 Format FS450_0001 64 Format FS450_0001 169 Format FS450_0007 46 GeneChip Whole Transcript Sense Target Labeling Assay Manual
20. centration of 1X loading dye Prepare 10 bp and 100 bp DNA ladders 1 uL ladder 7 uL water 2 uL loading dye for each lane Carefully load samples and two ladders on gel Each well can hold a maximum of 20 uL Run the gel at 150 volts until the front dye red almost reaches the bottom The electrophoresis takes approximately 1 hour While the gel is running prepare at least 100 mL of a 1X solution of SYBR Gold for staining 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 After the gel is complete break open cartridge and stain the gel in 1X SYBR Gold for 10 minutes Place the gel on the UV light box and produce an image following standard procedure Be sure to use the appropriate filter for SYBR Gold Fluidics Protocols And Fluidics Scripts for GeneChip ST Arrays Please refer to the GeneChip Expression Wash Stain and Scan User Manual P N 702731 for detailed information on the washing and staining steps required Fluidics protocols and fluidics scripts for GeneChip ST Arrays are provided below for your convenience This information is also available online at www affymetrix com Fluidics Protocols Table C 1 Fluidics Protocols for the GeneChip ST Arrays Fluidics Station 450 FS450_0001 and FS450_0007 Post Hyb Wash 1 10 cycles of 2 mixes cycle with Wash Buffer A at 30 C Post Hy
21. e Incubate at room temperature for 5 minutes Spin at 25 000 x g maximum speed for 1 minute Elute a second time by pipetting the flow through in the Collection Tube 713 5 uL back onto the Spin Column membrane Place the Spin Column back into the Collection Tube and incubate at room temperature for 5 minutes Spin at 25 000 x g maximum speed for 1 minute The eluted cRNA is 13 5 uL Determine the cRNA yield by spectrophotometric UV measurement at 260 nm 280 nm and 320 nm Concentration of cRNA ug uL A A329 x 0 04 x dilution factor ug of cRNA eluate in uL x concentration of cRNA in ug uL The NanoDrop ND 1000 can also be used to measure the concentration NOTE The average yield may vary depending on the type of tissue used and the quality of the RiboMinus rRNA reduction step NOTE Store eluted cRNA at 80 C if not proceeding immediately to Procedure E NOTE If cRNA concentration is too low to obtain 8 to 10 ug of cRNA in 6 5 pL cRNA can be concentrated using a SpeedVac 26 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure E Second Cycle First Strand cDNA Synthesis This Procedure requires the use of the GeneChip WT cDNA Synthesis Kit 1 Mix cRNA sample from Procedure D with the Random Primers in a strip tube as listed in Table 3 5 below Table 3 5 Second Cycle CRNA Random Primers Mix Component Volume in 1 Rxn cRNA 10 ug variab
22. eling 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 as shown in the gel photograph Figure B 1 The procedure takes approximately 90 minutes to complete x NOTE The absence of a shift pattern indicates poor biotin labeling The problem should be addressed before proceeding to the hybridization step Figure B 1 Gel Shift 1 2 3 4 5 6 Lane 1 10 bp DNA Ladder Lane 2 Fragmented and labeled HeLa RNA Lane 3 Fragmented and labeled HeLa RNA with Avidin Lane 4 Fragmented and labeled HeLa RNA Lane 5 Fragmented and labeled HeLa RNA with Avidin Lane 6 100 bp DNA Ladder 1 Prepare a NeutrAvidin solution of 2 mg mL in PBS Place a 4 to 20 TBE gel into the gel holder and load system with 1X TBE Buffer For each sample to be tested remove two 1 uL aliquots of fragmented and biotinylated sample to fresh tubes Heat the aliquots of samples at 70 C 2 minutes 4 Add 5 uL of 2 mg mL NeutrAvidin to one of the two tubes for each sample tested 44 GeneChip Whole Transcript Sense Target Labeling Assay Manual 10 11 12 Mix and incubate at room temperature for 5 minutes Add loading dye to all samples to a final con
23. ented Single Stranded DNA Table 3 9 Labeling Reaction Component Volume in 1 Rxn Fragmented Single Stranded DNA from Procedure G 45 uL 5X TdT Buffer 12 uL TdT 2 uL DNA Labeling Reagent 5 mM 1 uL Total Volume 60 uL 2 After adding the labeling reagents to the fragmented DNA samples flick mix and spin them down Incubate the reactions at u 37 C for 60 minutes 70 C for 10 minutes u 4 C for at least 2 minutes Remove 2 pL of each sample for Gel shift analysis optional as described in Appendix B to assess the labeling efficiency 32 GeneChip Whole Transcript Sense Target Labeling Assay Manual 100 ng Total RNA Labeling Protocol NOTE The 100 ng Total RNA Labeling Protocol is recommended to be used with the GeneChip Gene 1 0 ST Arrays For optimal sensitivity on the GeneChip Exon 1 0 ST Arrays the 1 yg Total RNA Labeling Protocol Chapter 2 should be followed Procedure A Preparation of dilutions of Poly A RNA Controls Follow the same procedure as described in Chapter 2 Procedure A on page 11 Procedure B Preparation of T7 N Primers Poly A RNA Controls 1 Prepare a fresh 250 ng uL T7 N Primers dilution from a 2 5 ug uL stock by adding the concentrated T7 N Primers to the diluted Poly A RNA controls using a non stick RNase free microfuge tube as follows Table 4 1 First cycle Primer Poly A RNA Controls Total RNA Volume Component 100 ng 300 ng T7 N
24. figurations of the WT Assay with key differences indicated in Table 1 1 The 1 ug Total RNA Labeling Protocol starts with a ribosomal RNA rRNA reduction procedure where the 28S and 18S rRNA population is significantly reduced from the total RNA sample minimizing the background and thereby increasing the array detection sensitivity and specificity The rRNA reduction becomes critical when a user is interested in high sensitivity analysis of expression levels for both genes and exons using the GeneChip Exon 1 0 ST Arrays This is because exon probe sets contain a smaller number of probes and in some cases selection of those probes is constrained by the limited size of the probe selection region Therefore it is imperative to use the high sensitivity assay for optimal performance The protocol has been optimized for the 1 ug input amount However a modest increase in cRNA yield has been observed by increasing the amount of total RNA used Input amounts up to 2 ug show no adverse impact on array performance However at 2 ug it is highly recommended to scale up the RiboMinus reagents to insure efficient rRNA reduction Failure to do so may have a small negative impact on sensitivity particularly for analyses at the exon level On the other hand analysis of the gene level benefits from a larger number of high quality probes selected from the entire transcript thus the advantage of the additional rRNA reduction step is reduced Therefore the 100 ng
25. hole Transcript Sense Target Labeling Assay Manual Procedure F Hydrolysis of cRNA and Cleanup of Single Stranded DNA This Procedure requires the use of the GeneChip WT cDNA Synthesis Kit and the GeneChip Sample Cleanup Module 1 10 11 Add 1 pL of RNase H to each of the samples and incubate at u 37 C for 45 minutes u 95 C for 5 minutes u 4 C for 2 minutes Proceed to the cleanup step using the cDNA Cleanup Spin Columns from the GeneChip Sample Cleanup Module following the protocol as described below Store the sample at 20 C if not purifying the Single Stranded DNA immediately If not already done add 24 mL of Ethanol 100 to the cDNA Wash Buffer supplied in the GeneChip Sample Cleanup Module Add 80 uL of RNase free water to each sample followed by 370 uL of cDNA Binding Buffer and vortex for 3 seconds Apply the entire sample the total volume is 471 uL to a cDNA Spin Column sitting in a 2 mL Collection Tube Spin at 2 8 000 x g for 1 minute Discard the flow through Transfer the cDNA Cleanup Spin Column to a new 2 mL Collection Tube and add 750 uL of cDNA Wash Buffer to the column Spin at gt 8 000 x g for 1 minute and discard the flow through Open the cap of the cDNA Cleanup Spin Column and spin at lt 25 000 x g for 5 minutes with the caps open Discard the flow through and place the column in a 1 5 mL collection tube Pipet 15 uL of the cDNA Elution Buffer directly to the column membrane a
26. illing the probe array cartridge one for filling and the second to allow venting of air from the hybridization chamber NOTE Ensure that the bubble inside the hyb chamber floats freely upon rotation to allow the hybridization cocktail to make contact with all portions of the array Figure 5 1 GeneChip Probe Array Plastic cartridge Front Probe array on glass substrate 6 Place array in 45 C hybridization oven at 60 rpm and incubate for 17 hours 1 hour During the latter part of the array hybridization commence preparation of the reagents required immediately after completion of hybridization Wash Stain and Scan Please refer to the GeneChip Expression Wash Stain and Scan User Manual P N 702731 for the washing and staining steps required immediately after completion of hybridization Please refer to Appendix C for fluidics protocols and fluidics script information for GeneChip ST Arrays FAQ WT Sense Target Labeling Assay 1 What is the basic principle of the ribosomal RNA reduction procedure using the RiboMinus Human Mouse Transcriptome Isolation Kit Four biotinylated LNA RiboMinus probes are designed to specifically bind to the abundant 18S and 28S rRNA species 2 probes each for 18S and 28S rRNA Following hybridization of the biotinylated probes to the rRNA molecules in the total RNA sample the rRNA is efficiently removed from the sample by the addition of the RiboMinus Magnetic
27. imal transition to the target labeling process immediately following the rRNA reduction steps 1 Prepare the buffer by mixing these components as listed in Table 2 2 Keep tube at room temperature Table 2 2 Hybridization Buffer with Betaine Starting Total RNA Amount 1 ug 2 ug Component Volume for 1 Rxn Volume for 1 Rxn Betaine 5M 54 uL 84 uL Invitrogen Hybridization buffer 126 uL 196 uL Total Volume 180 uL 280 uL A 30 uL overfill is included in the Total Volume sufficient for completing the RiboMinus procedure for a single total RNA sample 14 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure C RiboMinus Probe Hybridization This Procedure requires the use of the RiboMinus Human Mouse Transcriptome Isolation Kit that needs to be obtained directly from Invitrogen 1 In a 0 2 mL strip tube mix the following components in Table 2 3 Keep the tubes on ice If the total RNA is in 1 ug uL of concentration follow the instructions in the second column If the total RNA sample is at a lower concentration of between 0 31 ug uL to 1 ug uL then follow the protocol listed in the third column of Table 2 3 lt a NOTE It is recommended to prepare a master mix including the RiboMinus probe and the Hybridization Buffer with Betaine and then add the combined amount see Table 2 3 to the tube containing the Total RNA Poly A RNA Control Mix Chapter 2 rRNA Reduction and Prepara
28. ing Assay Manual Procedure D Preparation of Beads This Procedure requires the use of the RiboMinus Human Mouse Transcriptome Isolation Kit that needs to be obtained directly from Invitrogen NOTE Procedures D and E require the use of a 37 C heat block and a 50 C heat block Pre heat the heat blocks prior to initiating these steps A heating block at 37 C is required Use the volumes between parenthesis if the starting amount of total RNA is 2 ug 1 Completely re suspend the bottle containing magnetic beads by flicking it until no deposit is observed at the bottom of the bottle Pipet 50 uL 100 uL of beads suspension into a 1 5 mL non stick RNase free tube Steps 3 to 6 are done at room temperature Briefly spin and place the tube with the beads suspension on the magnetic stand for 1 minute With the tube remaining in the stand gently aspirate and discard the supernatant NOTE Drying of the beads decreases the bead efficiency therefore ensure that the beads are hydrated at all times It is recommended to handle a limited number of tubes at one time to reduce the risk of drying the beads 4 1st Wash A Add 50 uL 100 uL of RNase free water to the beads and re suspend them by flicking the tube NOTE If the beads are still attached to the wall of the tube put the tube back into the magnetic stand and rotate the tube with quick motion until the beads are in suspension B Spin briefly Place
29. ion Fragmented and Labeled DNA 60 0 uL 27 uL 25 ng uL Target from Chapter 3 Control Oligonucleotide B2 3 nM 3 7 uL 1 7 uL 50 pM 20X Eukaryotic Hybridization 11 uL 5 uL 1 5 5 25 and Controls bioB bioC bioD cre 100 pM respectively 2X Hybridization Mix 110 uL 50 uL 1X DMSO 15 4 uL 7 uL 7 Nuclease free Water up to 220 0 uL up to 100 Total Volume 220 0 uL 100 uL This volume is 58 uL if a portion of the sample was set aside for Gel shift analysis 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 cRNA before aliquoting Flick or gently vortex the tubes and spin down Heat the Hybridization Cocktail at 99 C for 5 minutes Cool to 45 C for 5 minutes and centrifuge at maximum speed for 1 minute 4 Equilibrate the GeneChip ST Array to room temperature immediately before use Label the array with the name of the sample that will be hybridized 36 GeneChip Whole Transcript Sense Target Labeling Assay Manual 5 Inject the appropriate amount see Table 5 2 of the specific sample into the array through one of the septa see Figure 5 1 for location of the septa on the array Table 5 2 Probe Array Cartridge Volumes for Hybridization Cocktail Array Format Volume 49 Standard 200 uL 64 200 uL 169 80 uL NOTE It is necessary to use two pipette tips when f
30. karyotic Total RNA Nano Assay in the Bioanalyzer Please see the Reagent Kit Guide provided with the RNA 6000 Nano LabChip Kit for instructions Based on the Bioanalyzer results on average 7100 ng of rRNA reduced sample may be recovered from 1 ug of total RNA starting material but the recovery rate may vary depending on the tissue type See Figure 2 1 for an example of the results 13 NOTE It is recommended to analyze 100 ng 1 pL of total RNA sample without RiboMinus Kit treatment as a control Figure 2 1 Bioanalyzer profile of Human Brain total RNA before and after rRNA reduction with RiboMinus Kit treated samples are in triplicates Starting Total RNA Fluorescence rRNA Reduced Total RNA Poly A RNA Controls Mix Migration Time 1 pg Total RNA Target Labeling Protocol NOTE The 1 ug Total RNA Labeling Protocol is recommended for use with GeneChip Exon 1 0 ST Arrays To prepare targets for GeneChip Gene 1 0 ST Arrays follow the 100 ng Total RNA Labeling Protocol in Chapter 4 Procedure A Preparation of rRNA Reduced Total RNA Poly A RNA Controls T7 N Primers Mix This Procedure requires the use of the GeneChip WT cDNA Synthesis Kit 1 Dilute the T7 N Primers 2 5 ug uL stock 1 5 with RNase free water to make up a 500 ng uL working solution Keep on ice Prepare fresh every time 2 Mix the diluted T7 N Primers with the Concentrated rRNA Reduced Sample from Chapter 2 P
31. le Random Primers 3 ug uL 1 5 uL RNase free water up to 8 uL Total Volume 8 0 uL For some samples that generate high yield in the second cycle cDNA synthesis reaction 8 ug may be used to obtain 5 ug of single stranded DNA target The starting amount of cRNA varies depending on the type of tissue used Flick mix and spin down the tubes Incubate the Second Cycle cCRNA Random Primers Mix at u 70 C for 5 minutes u 25 C for 5 minutes Cool the samples at 4 C for at least 2 minutes 5 Ina separate tube prepare the Second Cycle Reverse Transcription Master Mix as described in Table 3 6 Add the SuperScript II enzyme to the master mix last and proceed immediately to aliquot into tubes from Step 4 as described in Step 6 on page 27 Table 3 6 Second Cycle First Strand cDNA Synthesis Master Mix Component Volume in 1 Rxn 5X 1 Strand Buffer 4 0 uL DTT 0 1M 2 0 uL dNTP dUTP 10 mM 1 25 uL SuperScript II 4 75 uL Total Volume 12 0 uL Chapter 3 1 ug Total RNA Target Labeling Protocol 27 Transfer 12 uL of the Second Cycle First Strand cDNA Synthesis Master Mix to the Second Cycle cRNA Random Primers Mix from Procedure E Step 4 for a total reaction volume of 20 uL Mix thoroughly by gently flicking the tubes a few times and centrifuge briefly Incubate the reactions at u 25 C for 10 minutes 42 C for 90 minutes u 70 C for 10 minutes u 4 C for at least 2 minutes 28 GeneChip W
32. minutes u 70 C for 10 minutes 4 Cool the reaction to 4 C for at least 2 minutes before immediately continuing to the First Cycle Second Strand cDNA Synthesis f NOTE Keeping the reaction at 4 C longer than 10 minutes may result in reduced cRNA yields Chapter 3 1 ug Total RNA Target Labeling Protocol 23 Procedure C First Cycle Second Strand cDNA Synthesis This Procedure requires the use of the GeneChip WT cDNA Synthesis Kit 1 Make a fresh dilution of 17 5 mM MgCl each time Mix 2 uL of IM MgCl with 112 uL of RNase free water Prepare the First Cycle Second Strand Master Mix as described in Table 3 3 Add the RNase H and DNA Polymerase I enzymes to the master mix last and proceed immediately to aliquot into the tubes from Procedure B Step 4 as described in Step 3 below Table 3 3 First Cycle Second Strand Master Mix Component Volume in 1 Rxn RNase free Water 4 8 uL MgCl 17 5 mM 4 0 uL dNTP Mix 10 mM 0 4 uL DNA Polymerase I 0 6 uL RNase H 0 2 uL Total Volume 10 0 uL 3 Add 10 uL of the First Cycle Second Strand Master Mix to the reaction tube from the First Strand cDNA Synthesis Reaction in Procedure B for a total reaction volume of 20 uL Flick or gently vortex the tubes and spin down Incubate the reaction in a thermal cycler at 16 C for 120 minutes without heated lid a 75 C for 10 minutes with heated lid Cool the sample for at least 2 minutes at 4 C before immedi
33. munoPure NeutrAvidin Pierce 31000 PBS pH 7 2 Invitrogen 20012 027 Or equivalent 8 GeneChip Whole Transcript Sense Target Labeling Assay Manual Miscellaneous Supplies Table 1 4 Miscellaneous Supplies Materials Source P N Miscellaneous Supplies 1 5 mL RNase free Microfuge Tubes Ambion 12400 1 5 mL Non stick RNase free Microfuge Tubes Ambion 12450 0 2 mL MicroAmp reaction tubes 8 tubes strip Applied Biosystems N801 0580 MicroAmp caps for 8 strip tubes Applied Biosystems N801 0535 Pipette for 25 mL VWR 53283 710 Pipet aid VWR 53498 103 Tough Spots USA Scientific 9185 Or equivalent Instruments Table 1 5 Instruments Chapter 1 Overview 9 Instruments NanoDrop ND 1000 Manufacturer NanoDrop Technologies P N N A Barcode Reader Optional GeneChip Hybridization Oven 640 Affymetrix 800138 110 v 800139 220 v Eppendorf Centrifuge Eppendorf 5417C Tube Strip Picofuge Stratagene 400540 PicoFuge Stratagene 400550 GeneChip Fluidics Station 450 Affymetrix 00 0079 GeneChip Scanner 3000 7G Affymetrix 00 0212 North America 00 0213 International GeneChip AutoLoader with External Affymetrix 00 0090 GCS 3000 7G S N 501 00 0129 GCS 3000 7G S N 502 ABI GeneAmp PCR System 9700 Applied Biosystems N8050001 Bioanalyzer 2100 Agilent G2940CA Heating blocks VWR 13259 030 Pipette for 0 1 to 2 uL Rai
34. nd incubate at room temperature for 1 minute Then spin at 25 000 x g for 1 minute Repeat the elution step by pipetting another 15 uL of the cDNA Elution Buffer directly to the column membrane and incubate at room temperature for 1 minute Then spin at 25 000 x g for 1 minute The total volume of the eluted Single Stranded DNA is 28 uL total Take 2 uL from each sample to determine the yield by spectrophotometric UV measurement at 260 nm 280 nm and 320 nm Concentration of Single Stranded DNA ug uL A A359 x 0 033 x dilution factor ug of DNA eluate in uL x concentration of DNA in ug uL Each tube should have 2 5 5 ug of Single Stranded DNA The NanoDrop ND 1000 can also be used to measure the concentration NOTE Eluted single stranded cDNA can be stored overnight at 20 C if not proceeding immediately to Procedure G Chapter 3 1 ug Total RNA Target Labeling Protocol 29 Procedure G Fragmentation of Single Stranded DNA This Procedure requires the use of the GeneChip WT Terminal Labeling Kit 1 Set up fragmentation reaction in 0 2 mL strip tubes using Table 3 7 Table 3 7 Fragmentation Master Mix Component Volume Amount in 1 Rxn Single Stranded DNA 5 5 ug RNase free Water up to 31 2 uL Total Volume 31 2 uL 2 Prepare the Fragmentation Master Mix using Table 3 8 Table 3 8 Fragmentation Master Mix Component Volume in 1 Rxn RNase free Water 10 uL 10X cD
35. nds 11 Procedure A Preparation of Dilutions of Poly A RNA Controls 11 Procedure B Preparation of Hybridization Buffer with Betaine 13 Procedure C RiboMinus Probe Hybridization 5 14 Procedure D Preparation of Beads WW kk KK KK KK K R KK KRI 16 Procedure E rRNA Reduction Jk kk kk kk KK KK cece eee eee 18 Procedure F Concentration kk kk kk kk kK KK KK KK eee 19 Procedure G Analysis with Bioanalyzer 0 0 000002 20 1 ug Total RNA Target Labeling Protocol 21 Procedure A Preparation of rRNA Reduced Total RNA Poly A RNA Controls T7 N g Primers Mix a e RES ihr KK KK KK KK KK Seeds 21 Procedure B First Cycle First Strand cDNA Synthesis 22 Procedure C First Cycle Second Strand cDNA Synthesis 23 Procedure D First Cycle CRNA Synthesis and Cleanup 24 Procedure E Second Cycle First Strand cDNA Synthesis 26 Procedure F Hydrolysis of cRNA and Cleanup of Single Stranded DNA 28 Procedure G Fragmentation of Single Stranded DNA 29 Procedure H Labeling of Fragmented Single Stranded DNA 31 GeneChip Whole Transcript Sense Target Labeling Assay Manual Chapter 4 Chapter 5 AppendixA Appendix B Appendix C 100 ng Total RNA Labeling Protocol 2 33 Procedure A Preparation of dilutions of Poly A RNA Controls 33 Procedure B Preparation of T7 N
36. neChip Whole Transcript Sense Target Labeling Assay Manual 2 Add 2 uL of Poly A RNA Control Stock to 38 uL of Poly A Control Dil Buffer to make the First Dilution 1 20 3 Mix and spin to collect the solution at the bottom of the tube 4 Add 2 uL of the First Dilution to 98 uL of Poly A Control Dil Buffer to make the Second Dilution 1 50 Mix and spin to collect the solution at the bottom of the tube When using 1 ug of total RNA as starting material add 2 uL of the Second Dilution to 98 uL of Poly A Control Dil Buffer to make the Third Dilution 1 50 When using 2 ug of total RNA as starting material add 2 uL of the Second Dilution to 48 uL of Poly A Control Dil Buffer to make the Third Dilution 1 25 7 Mix and spin to collect the solution at the bottom of the tube Add 2 uL of the appropriate Third Dilution to 1 ug or 2 ug of total RNA to make up the Total RNA Poly A RNA Controls Mix as described in Table 2 1 Chapter 2 rRNA Reduction and Preparation of Total RNA with Diluted Poly A RNA Controls 13 Procedure B Preparation of Hybridization Buffer with Betaine This Procedure requires the use of the RiboMinus Human Mouse Transcriptome Isolation Kit that needs to be obtained directly from Invitrogen bu IMPORTANT The protocol for using the RiboMinus Kit for rRNA reduction for GeneChip arrays has been modified from the manufacturer s original recommendation Follow only the procedure described in this manual for opt
37. nin 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 Or equivalent 10 GeneChip Whole Transcript Sense Target Labeling Assay Manual Suggested Workflow Day 1 Complete Chapter 2 rRNA Reduction 1 5 hours for GeneChip Exon 1 0 ST Arrays Complete Chapter 3 or 4 Procedures A C First Cycle Synthesis of cDNA 3 5 hours Start Chapter 3 Procedure D First Cycle Synthesis of CRNA 16 hours Start on Day 1 finish on Day 2 Day 2 u Complete Chapter 3 Procedures D I Second Cycle Synthesis of first strand DNA fragmentation and labeling 8 hours u Start Chapter 5 Hybridization 17 hours Start on Day 2 finish on Day 3 Day 3 u Array Washing Staining and Scanning 2 hours Please refer to the GeneChip Expression Wash Stain and Scan User Manual P N 702731 Assumes that only one sample is carried through the assay The estimated time required may be longer if multiple samples are processed simultaneously rRNA Reduction and Preparation of Total RNA with Diluted Poly A RNA Controls f NOTE The rRNA reduction step may be omitted when following the 100 ng Total RNA Labeling Protocol to prepare targets for GeneChip Gene 1 0 ST Arrays Proceed directly to Chapter 4 and follow the 100 ng Total Labeling Protocol Procedure A Preparation of Dilutions of Poly A RNA Controls This Procedure requires
38. ons per kit will be 24 if using 2 ug of total RNA Table 1 2 Continued Necessary Reagents Chapter 1 Overview 5 Material Source P N GeneChip WT cDNA Synthesis and Amplification Kit Sub kit 1 GeneChip WT cDNA Synthesis Kit Contains n T7 N Primers 2 5 ug L n 5X 1 Strand Buffer a DTT 0 1M a dNTP 10 mM a RNase Inhibitor a SuperScript II n MgCL 1M o DNA Polymerase I o RNase H o Random Primers 3 ug uL o dNTP dUTP 10 mM o RNase free Water Sub kit 2 GeneChip WT cDNA Amplification Kit Contains a 10X IVT Buffer o IVT NTP Mix o IVT Enzyme Mix n IVT Control Affymetrix 900673 30 Rxn or 900672 10 Rxn Fragmentation and Labeling GeneChip WT Terminal Labeling Kit Contains o 10X cDNA Fragmentation Buffer a UDG 10 U uL o APE 1 1 000 U uL o 5X TdT Buffer o TdT 30 U uL o DNA Labeling Reagent 5 mM o RNase free Water Affymetrix 900671 30 Rxn or 900670 10 Rxn 6 GeneChip Whole Transcript Sense Target Labeling Assay Manual Table 1 2 Continued Necessary Reagents containing Hybridization Module from Box 1 a Pre Hybridization Mix o 2X Hybridization Mix a DMSO a Nuclease free water Stain Module from Box 1 o Stain Cocktail 1 o Stain Cocktail 2 a Array Holding Buffer Wash Buffers A and B from Box 2 a Wash Buffer A P N 900721 o Wash Buffer B P N 900722 Material Source P N cDNA cRNA Cleanup GeneChip IVT cR
39. ormed in oven incubators for even temperature distribution or in a thermal cycler Hold at 4 C 4 Proceed to the cleanup procedure for cRNA using the cRNA Cleanup Spin Columns from the GeneChip Sample Cleanup Module following the protocol described below Store the sample at 80 C if not purifying the cRNA immediately 5 Ifnot already done add 20 mL of Ethanol 100 to the CRNA Wash Buffer supplied in the GeneChip Sample Cleanup Module Add 50 uL of RNase free water to each IVT reaction to a final volume of 100 uL Add 350 uL of cRNA Binding Buffer to each sample and vortex for 3 seconds Add 250 uL of 100 ethanol to each reaction and flick mix Apply the sample to the IVT cRNA Cleanup Spin Column sitting in a 2 mL Collection Tube 10 Centrifuge for 15 seconds at 2 8 000 x g Discard the flow through o o Ng 11 12 13 14 15 16 Chapter 3 1 ug Total RNA Target Labeling Protocol 25 Transfer the IVT cRNA Cleanup Spin Column to a new 2 mL Collection Tube Add 500 uL of cRNA Wash Buffer to column and centrifuge for 15 seconds at 2 8 000 x g Discard the flow through Wash again with 500 uL of 80 v v Ethanol Centrifuge for 15 seconds at 2 8 000 x g and discard the flow through Open the column cap and spin at 25 000 x g maximum speed for 5 minutes with the caps open Transfer the IVT cRNA Cleanup Spin Column to a new 1 5 mL Collection Tube and add 15 uL of RNase free water directly to the membran
40. r the WT targets What Fluidics Protocol do I use for the GeneChip ST Arrays New Fluidics Protocols have been developed for this assay FS450_0001 for Exon Arrays and FS450 0007 for Gene Arrays In addition to tubes containing SAPE and anti streptavidin biotinylated antibody there is a tube containing 1X Array Holding Buffer which is added to the cartridge following the wash stain procedure Please refer to the GeneChip Whole Transcript Sense Target Labeling Assay Manual Chapter 5 for more details How long does it take to scan an array It takes approximately 35 minutes to scan each Exon Array and approximately 10 minutes to scan each Gene Array 21 22 Appendix A FAQ 41 What are the internal grid lines on the array image for There is a new gridding algorithm in GCOS 1 3 specifically designed for image analysis of arrays with 5 um or smaller feature size This algorithm utilizes 169 subgrids 13x13 to address probe cells on the array This method is superior to global gridding for arrays with feature pitch less than 11 um How do check for the correct gridding of the new array If any of the subgrids fail to align GCOS will fail to generate a cel file The failed subgrids will be visualized with red borders and an x in the center rather than white borders for successfully aligned subgrids 42 GeneChip Whole Transcript Sense Target Labeling Assay Manual Gel Shift Assay The efficiency of the lab
41. rocedure F as listed in Table 3 1 using 0 2 mL strip tubes on ice Table 3 1 First cycle Preparation of rRNA reduced Total RNA Poly A RNA Controls T7 N Primers Component Volume in 1 Rxn rRNA Reduced Total RNA Poly A RNA Controls Mix 4 uL Diluted T7 N Primers 500 ng uL 1 uL Total Volume 5 uL 3 Flick the tube to mix spin down the tube and incubate at 70 C for 5 minutes u 4 C for at least 2 minutes 4 Spin down and place on ice for use in Procedure B 22 GeneChip Whole Transcript Sense Target Labeling Assay Manual Procedure B First Cycle First Strand cDNA Synthesis This Procedure requires the use of the GeneChip WT cDNA Synthesis Kit 1 Prepare the First Cycle First Strand Master Mix as shown in Table 3 2 Add the SuperScript II enzyme to the master mix last and proceed immediately to aliquot into the tubes from Procedure A Step 4 as described in Step 2 below Table 3 2 First Cycle First Strand Master Mix Component Volume in 1 Rxn 5X 1st Strand Buffer 2 uL DTT 0 1M 1 uL dNTP Mix 10 mM 0 5 uL RNase Inhibitor 0 5 uL SuperScript II 1 uL Total Volume 5 uL 2 Add 5 uL of the First Cycle First Strand Master Mix to the tube containing the Concentrated rRNA Reduced Total RNA Poly A RNA Controls T7 N Primers Mix from Procedure A flick mix and spin down The total reaction volume is 10 uL 3 Incubate the reaction at u 25 C for 10 minutes u 42 C for 60
42. rotocol Day 1 Day 2 r Day 3 Assume that only one sample is carried through the assay The time may be longer if multiple samples are processed simultaneously RiboMinus Human Mouse Transcriptome Isolation Kit is required for this step and needs to be ordered directly from Invitrogen 4 GeneChip Whole Transcript Sense Target Labeling Assay Manual Materials Necessary Reagents Table 1 2 Necessary Reagents Material Source P N rRNA Reduction Not required for the 100 ng Total RNA Labeling Protocol RiboMinus Transcriptome Isolation Kit Human Mouse Invitrogen K1550 02 49 Rxn Magna Sep Magnetic Particle Separator Invitrogen K1585 01 Betaine 5M Sigma Aldrich B 0300 Target Labeling GeneChip WT Sense Target Labeling and Control Reagents Affymetrix 900652 30 Rxn Contains one of each of the following kits that can also be ordered individually o GeneChip Eukaryotic Poly A RNA Control Kit 100 Rxn a GeneChip WT cDNA Synthesis and Amplification Kit 30 Rxn a GeneChip WT Terminal Labeling Kit 30 Rxn a GeneChip Sample Cleanup Module 30 Rxn a GeneChip IVT cRNA Cleanup Kit 30 Rxn a GeneChip Hybridization Control Kit 30 Rxn cDNA Synthesis and Amplification GeneChip Eukaryotic Poly A RNA Control Kit Affymetrix 900433 Contains 100 Rxn o Poly A Control Stock a Poly A Control Dil Buffer 49 reactions when using 1 ug of total RNA The number of reacti
43. should have a peak centered around 40 to 70 bases with the majority of the fragments ranging from 20 bases to 200 bases Are there any safe stopping points in the assay What are recommended storage conditions There are a few safe stopping points in the assay including After RNA Clean up Concentration following the RiboMinus rRNA reduction step before proceeding to first cycle first strand cDNA synthesis Store rRNA reduced total RNA at 80 C u After IVT reaction and the cRNA cleanup step in the first cycle before proceeding to the second cycle of reverse transcription Store cRNA at 80 C a After reverse transcription and the single stranded cDNA cleanup step in the second cycle before fragmentation and labeling Store single stranded cDNA at 20 C u After fragmentation and labeling before hybridization Store labeled cDNA at 20 C How much single stranded DNA target do you need to hybridize to one array It is recommended to hybridize approximately 5 ug or 2 ug of fragmented and labeled DNA target to each Exon or Gene Arrays respectively What is the hybridization condition As described in the GeneChip Whole Transcript Sense Target Labeling Assay Manual a final concentration of 7 DMSO is included in the hybridization cocktail for hybridizing the WT sense target to ST arrays 40 GeneChip Whole Transcript Sense Target Labeling Assay Manual 15 16 17 Can I hybridize the DNA target to the
44. the tube on the magnetic stand for 1 minute With the tube remaining in the stand gently aspirate and discard the supernatant 2nd Wash Add 50 uL 100 uL of RNase free water to the beads and re suspend them by flicking the tube Place the tube on the magnetic stand for 1 minute With the tube remaining in the stand gently aspirate and discard the supernatant Chapter 2 rRNA Reduction and Preparation of Total RNA with Diluted Poly A RNA Controls 17 3rd Wash Add 50 uL 100 uL of the Hybridization Buffer with Betaine from Procedure B to the beads and re suspend them by flicking the tube Spin briefly Place the tube on the magnetic stand for 1 minute With the tube remaining in the stand gently aspirate and discard the supernatant NOTE When the Hybridization Buffer with Betaine is added in the 3rd Wash even after complete settling of the beads on the magnetic stand a thin film of beads may coat the entire inside of the tube so the solution may appear yellow Carefully aspirate out the supernatant from the center of the tube which should be clear in color 7 Re suspend the beads in Hybridization Buffer with Betaine Keep them at 37 C ina heating block for to 2 minutes Refer to Table 2 3 for appropriate volume NOTE If there is a large number of samples to be processed at the same time the beads can be prepared in a large batch This can be done with up to a total of 10 samples by increasing the volume of the
45. tion of Total RNA with Diluted Poly A RNA Controls 15 Table 2 3 RiboMinus Reaction and Wash Volumes Total RNA Amount Multiple applications to column required column capacity x 800 uL RNA Concentration Range 1 ug uL 0 31 ug pL to 1 ug uL 20 62 ug uL RiboMinus Probe Hybridization Total RNA Poly A Controls Mix from Procedure A 3 0 uL up to 5 2 uL up to 5 2 uL RiboMinus Probe 100 pmol uL 0 8 uL 0 8 uL 1 6 uL Hybridization Buffer with Betaine from Procedure B 20 uL 30 uL 40 uL Total Hybridization Volume 23 8 pL 36 0 pL 46 8 pL Beads Preparation Magnetic Beads 50 uL 50 uL 100 uL 2X Wash with Water 50 uL 50 uL 100 uL 1X Wash with Hybridization Buffer with Betaine 50 uL 50 uL 100 uL Re suspension of Beads with Hybridization Buffer 30 uL 20 uL 60 uL with Betaine Final Wash of Beads with Hybridization Buffer with 50 uL 50 uL 50 uL Betaine Concentration and Clean up Approximate Supernatant Volume 100 pL 100 pL 150 pL cRNA Binding Buffer 350 uL 350 uL 525 uL 10096 Ethanol 250 uL 250 uL 375 uL Total Volume Applied to Column 700 pL 700 pL 1 050 pL cRNA Wash Buffer 500 uL 500 uL 500 uL 8096 Ethanol 500 uL 500 uL 500 uL 2 Flick the tube gently to mix spin briefly and incubate at 70 C for 5 minutes in a thermal cycler 3 Quench the reaction immediately by placing the tube on ice while preparing the magnetic beads 16 GeneChip Whole Transcript Sense Target Label
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