User Manual, GeneAtlas 3' IVT Express Kit
Contents
1. Component Volume Storage BOX 1 of 2 Hybridization and Stain Components 1X Pre Hybridization Mix 9 0 mL 2 C to 8 C 1 3X Hybridization Mix Solution A 3 0 mL 2 C to 8 C 1 3X Hybridization Mix Solution B 5 6 mL 2 C to 8 C Nuclease free Water 2 0 mL 2 C to 8 C Stain Cocktail 1 52 5 mL 2 C to 8 C Stain Cocktail 2 26 3 mL 2 C to 8 C Array Holding Buffer 15 mL 2 C to 8 C BOX 2 of 2 Wash Buffers A and B Wash Buffer A 519 mL 2 C to 8 C Wash Buffer B 64 mL 2 C to 8 C 10 GeneAtlas 3 IVT Express Kit User Manual Materials Required Reagents Table 1 5 Reagents Material Source P N GeneAtlas 3 IVT Express Kit Affymetrix See Table 1 3 for detailed kit information 901649 20 Rxn Or equivalent t For optional analysis of aRNA size Instruments Table 1 6 Instruments GeneAtlas Hybridization Wash and Stain Kit for 3 IVT Arrays Affymetrix 901531 60 Rxn See Table 1 4 for detailed kit information 100 ethanol ACS reagent grade multiple Agilent RNA 6000 Nano Kitt Agilent Technologies 5067 1511 Instruments Manufacturer P N GeneAtlas Fluidics Station Affymetrix 00 0377 GeneAtlas Imaging Station Affymetrix 00 0376 GeneAtlas Hybridization Station Affymetrix 00 0380 115VAC 00 0381 230VAC GeneAtlas Workstation Affymetrix 00 0894 GeneAtlas Barcode Scanner Affymetrix 00 0379 Lab Equipment and Sup2. 4 times Centrifuge briefly to collect the reaction at the bottom of the tube plate and place on ice Place the reaction in a 16 C thermal cycler block It is important to pre cool the thermal cycler block to 16 C because subjecting the reaction to temperatures gt 16 C will compromise aRNA yield 3 Incubation A Incubate for 1 hour at 16 C followed by 10 minutes at 65 C in a thermal cycler using the program for Second Strand cDNA Synthesis Table 2 1 on page 13 NOTE Cover reactions with the heated lid of the thermal cycler even if its temperature cannot be adjusted to match the block temperature After the incubation centrifuge briefly 5 seconds to collect the double stranded cDNA at the bottom of the tube plate Place on ice and immediately proceed to the IVT below or freeze at 20 C E TIP STOPPING POINT Samples can be stored overnight at 20 C at this point if desired 20 GeneAtlas 3 IVT Express Kit User Manual In Vitro Transcription to Synthesize Labeled aRNA Reagents and Materials Required Table 2 10 Reagents and Materials Required Item Needed GeneAtlas 3 IVT Express Kit Box 2 IVT Biotin Label IVT Labeling Buffer IVT Enzyme Mix User supplied Reagents and Materials RNase free Microfuge Tubes for making master mix 1 Assembly of IVT Master Mix NOTE This step is performed at room temperature A Prepare an IVT Ma
3. Figure 4 1 appears KGS Affymetrix REGISTRATION No Strips Added 3 Click the Strip button edi The Add Strip Window appears Figure 4 2 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 4 3 Contents 35 KGS Affymetrix REGISTRATION Import Data Save and Proceed gt gt Sample File Name Probe Array Type Probe Array Position text BRE a SEEN 5 Under the Sample File 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 LE The Save dialog box appears Figure 4 4 CAPS Data Default Arr Folder I Date I 05 26 2009 14 31 13 Excel 05 26 2009 14 31 13 Protocol 05 26 2009 14 31 13 Template 05 26 2009 14 31 13 Workflow 05 26 2009 14 31 13 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 4 5 36 GeneAtlas 3 IVT Express Kit User Manual Figure 4 5 Sample Files Created as cr 8 Click OK NOTE You may enter a total of four array strips during the registration process To add additional
4. Other Important Parameters Keep reaction incubation times precise and consistent The incubation times for the enzymatic reactions in the protocol were optimized in conjunction with the kit reagents for maximum yield in each step adhere to them closely Use master mixes We strongly recommend preparing master mixes for each step of the GeneAtlas 3 IVT Express procedure This reduces the effects of pipetting error saves time and improves reproducibility The fill volumes in the kit allow for a 5 overage when making master mixes Mix each kit component before use u Mix enzyme solutions by gently flicking the tube a few times before adding them to master mixes o Thaw frozen reagents completely at room temperature then mix thoroughly by vortexing and place on ice Incubate reactions in a calibrated thermal cycler a We do not recommend using ordinary laboratory heat blocks water baths or hybridization ovens for any of the reaction incubations u The procedure is very sensitive to temperature therefore use a thermal cycler that has been calibrated according to the manufacturer s recommended schedule Variable or inaccurate incubation temperatures can negatively impact aRNA synthesis o Heated lids It is important that condensation does not form in the tubes during any ofthe incubations because it would change the reaction composition and can greatly reduce yield If possible set the lid temperature to match t
5. 776 6 291 183 6 346 413 6 399 365 6 610 482 6 733 977 and other U S or foreign patents Products are manufactured and sold under license from OGT under 5 700 637 and 6 054 270 Scanner Products may be covered by one or more of the following patents U S Patent Nos 6 141 096 6 262 838 6 294 327 6 403 320 6 407 858 6 597 000 7 406 391 and other U S or foreign patents Software Products may be covered by one or more of the following patents Products may be protected by one or more of the following patents U S Patent Nos 6 090 555 6 611 767 6 687 692 6 829 376 7 130 458 7 451 047 and other U S or foreign patents Copyright 2009 2010 Affymetrix Inc All rights reserved Contents Chapter 1 OVervieu cick cies ccteee ee etek e E hues E gn a nannies be 1 Chapter 2 Chapter 3 oru 222g e eae 4 eee an eee ee ne ae es eee 1 ASSAY OVENIEW ius uoce e Ghee pee ne e BUR REE oA dul IURE RE 2 Important Parameters for Successful Amplification 4 Other Important Parameters 6 Kit Contents and Storage Conditions 8 Materials SSSR 10 Required Reagents 10 Instruments 10 Lab Equipment and Supplies 11 aRNA Amplification Protocol 13 Equipment and Reagent Preparation 1
6. Affymetrix Array Strips on page 38 Target Hybridization Setup for Affymetrix Array Strips This section provides instruction for setting up array hybridizations using the GeneAtlas Hybridization Wash and Stain Kit for 3 IVT Arrays 60 rxns For ordering information please refer to Table 1 5 on page 10 Table 4 1 lists the necessary amount of aRNA required a single array These preparations take into account that it is necessary to make extra hybridization cocktail due to a small loss of volume 10 20 uL during each hybridization 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 two 2 hybridization trays per array strip C Set the temperature of the GeneAtlas Hybridization Station to 45 C D Warm the pre hybridization buffer to room temperature NOTE Aliquot 500 pL of pre hybridization buffer per array strip to be hybridized into an Eppendorf tube to accelerate the equilibration to room temperature Contents 39 2 Prepare the hybridization cocktail for each sample in an Eppendorf tube A Prepare sufficient hybridization cocktail Master Mix for all samples Table 4 1 Table 4 1 Hybridization Cocktail Setup Component Amount for 1 Array Master Mix for Final Dilution with 4 Arrays Fragmented and Includes 5 Overage Labeled aRNA Control Oligonucleo
7. beginning of this chapter but skip Step 2 of Target Hybridization Setup for Affymetrix Array Strips on page 38 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 of the four samples on the array strip Additional information includes Probe Array Type and Probe Array position Hybridization Software Setup During the Hybridization 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 34 GeneAtlas 3 IVT Express Kit User Manual 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 2 Click the Registration tab
8. strips please repeat Step 3 through Step 8 9 Proceed to Hybridization Software Setup on page 37 Contents 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 34 for instruction on registering array strips EH IMPORTANT When hybridizing more than one array strip per day it is recommended to keep the hybridization time consistent Set up hybridizations for one array strip at a time staggered by 1 to 1 5 hours so that washing and staining can occur immediately after completion of hybridization for each array strip the next day 1 Click the Hybridization tab on the GeneAtlas Software interface KG Affymetrix HYBRIDIZATION Strip 2 Click the Strip button adl The Add Strip Window appears Figure 4 7 Add Strip Scan or enter the bar code Bar Code Strip Name Time 30 Temperature 48 C aco 38 GeneAtlas 3 IVT Express Kit User Manual 3 Scan or enter the Bar Code required of an array strip you have registered The Strip Name field is automatically populated From the Instrument drop down box select the correct hybridization station 5 The Time and Temperature settings are automatically populated and are read from the installed library files 6 DO NOT click Start Proceed to Target Hybridization Setup for
9. supplied Reagents and Materials RNA Sample RNase free Microfuge Tubes for making master mix 1 Assembly of First Strand Master Mix A Thaw first strand synthesis reagents and place on ice B On ice assemble First Strand Master Mix in a nuclease free tube in the order listed in Table 2 7 Include 5 overage to cover pipetting error Table 2 7 First Strand Master Mix Component Amount Used Master Mix for for 4 Samples 1 Sample Includes 5 Overage First Strand Buffer Mix 2 uL 8 4 uL First Strand Enzyme Mix 0 5 uL 2 1 uL Total Volume 2 5 pL 10 5 uL C Mix well by gently vortexing Centrifuge briefly 5 seconds to collect the mix at the bottom of the tube D Place the supplied PCR Tubes or Plate on ice and transfer 2 5 uL First Strand Master Mix to individual tubes or wells 2 Addition of Total RNA poly A Control Mixture A Add 2 5 uL of the Total RNA poly A Control Mixture Table 2 5 to each aliquot of First Strand Master Mix for a final volume of 5 uL B Mix thoroughly by gently vortexing Centrifuge briefly to collect the reaction at the bottom of the tube plate and place on ice 18 GeneAtlas 3 IVT Express Kit User Manual 3 Incubation A Incubate for 2 hours at 42 C in a thermal cycler using the program for First Strand cDNA Synthesis Table 2 1 on page 13 E TIP When there is approximately 15 minutes left on the thermal cycler you may start reagent pre
10. 3 Prepare aRNA Wash Solution 13 Program the Thermal Cycler 13 Prepare Poly A RNA Controls 14 Reverse Transcription to Synthesize First Strand CDNA 17 Second Strand cDNA Synthesis 18 In Vitro Transcription to Synthesize Labeled aRNA 20 aRNA P tificatiOni 22 6202 mo de ees sie su Ecoute te 22 Evaluation and Fragmentation of aRNA 27 aRNA Quantitation and Expected Yield 27 Analysis of aRNA Size Optional 29 Fragmentation of Labeled aRNA 30 GeneAtlas 3 IVT Express Kit User Manual Chapter 4 Appendix A Appendix B Appendix C Hybridization 5444 dow ed in ee E OR we 33 GeneAtlas Software Setup 33 Sample Registration ice eb ew Edd d eae E dt 34 Hybridization Software Setup 37 Target Hybridization Setup for Affymetrix Array Strips 38 Hybridization of Array Strips on the GeneAtlas System 46 Rehybridizing Used Cocktails 48 Troubleshooting isse oe vase cu dn te eee or m Rx RR eon e 49 Positive Control Reaction 49 Control RNA Amplification Instructions 49 Expec
11. A Sample Key 9 Thyroid E Pancreas I MAQC B Brain B Heart I HeLa Cell Line Average aRNA Yield ug Total RNA Input Concentrate the purified aRNA Optional If necessary concentrate the aRNA by vacuum centrifugation If the heater on the vacuum centrifuge has different settings use medium or low Check the progress of drying every 5 10 minutes and remove the sample from the concentrator when it reaches the desired volume Avoid drying aRNA samples to completion Contents 29 Analysis of aRNA Size Optional The size distribution of aRNA can be evaluated using an Agilent 2100 Bioanalyzer with the Agilent RNA 6000 Nano Kit P N 5067 1511 or by conventional denaturing agarose gel analysis The bioanalyzer can provide a fast and accurate size distribution profile of aRNA samples but aRNA yield should be determined by UV absorbance or RiboGreen analysis To analyze aRNA size using a bioanalyzer follow the manufacturer s instructions for running the assay using purified aRNA Expected aRNA Size We recommend analyzing aRNA size distribution using an Agilent Bioanalyzer and RNA 6000 Nano Kit loaded with 300 ng of aRNA per well The expected aRNA profile is a distribution of sizes from 250 5500 nt with most of the aRNA between 600 1200 nt Average aRNA size may vary slightly depending on RNA quality and total RNA input amount Figure 3 2 Example Agilent Bioanalyzer Electropherogram of un fr
12. AX v Affymetrix User Manual GeneAtlas 3 IVT Express Kit For research use only Not for use in diagnostic procedures Trademarks Affymetrix GeneChip NetAffx Command Console Powered by Affymetrix GeneChip compatible Genotyping Console DMET GeneTitan Axiom and GeneAtlas are trademarks or registered trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Reagents Products may be protected by one or more of the following patents U S Patent Nos 6 864 059 7 468 243 7 491 818 Arrays Products may be covered by one or more of the following patents U S Patent Nos 5 445 934 5 744 305 6 261
13. N AM10050 ey Magnetic Stand aRNA Binding Step After addition of ethanol and mixing RNA Binding Beads Capture After 5 minutes on magnetic stand 52 GeneAtlas 3 IVT Express Kit User Manual Ambion Magnetic Stand 96 Ambion 96 well Magnetic Ring Stand P N AM10027 P N AM10050 Bead Washing After second wash and 1 minute shake Removal of Ethanol Dry beads following 1 minute shake aRNA Elution Dispersed beads following 3 minute shake Elution Step Recovery of purified aRNA following bead capture Shaker Speeds Table C 1 Plate Shaking Speeds aRNA Binding Gentle 300 500 4 1 Bead Washing Moderate 700 900 7 4 Ethanol Removal Vigorous 1000 1200 10 7 aRNA Elution Vigorous 1000 1200 10 7 1200 1000 oo e o Approximate RPM a e e LE A e o 200 0 1 2 3 4 5 6 7 8 9 10 Speed Setting 54 GeneAtlas 3 IVT Express Kit User Manual
14. NA sample will be amplified using GeneAtlas 3 IVT Express Kit RNA samples should be free of contaminating proteins DNA and other cellular material as well as phenol ethanol and salts associated with RNA isolation procedures Impurities can lower the Contents 5 efficiency of reverse transcription and subsequently reduce the level of amplification An effective measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm The ratio of A to A values should fall in the range of 1 7 2 1 RNA must be suspended in high quality water TE 10 mM Tris HCl 1 mM EDTA RNA Integrity The integrity of the RNA sample or the proportion that is full length is another important component of RNA quality Reverse transcribing partially degraded mRNAs will generate cDNAs that may lack portions of the transcripts that are interrogated by probes on the array RNA integrity can be evaluated by microfluidic analysis using the Agilent 2100 bioanalyzer with an RNA LabChip Kit Primarily full length RNA will exhibit a ratio of 28S to 18S rRNA bands that approaches 2 1 Using a bioanalyzer the RIN RNA Integrity Number can be calculated to further evaluate RNA integrity The RIN a metric developed by Agilent includes information from both the rRNA bands and outside the rRNA peaks potential degradation products to provide a picture of RNA degradation states Search for RIN at the following web site for further information www chem a
15. agmented aRNA generated from 50 ng of HeLa total RNA FU Unfragmented aRNA 35 304 254 204 25 200 500 1000 2000 4000 nt 30 GeneAtlas 3 IVT Express Kit User Manual Fragmentation of Labeled aRNA Reagents and Materials Required Table 3 1 Reagents and Materials Required Location Item Needed GeneAtlas 3 IVT Express Kit Box 1 Nuclease free Water 5X Array Fragmentation Buffer 8 Strip PCR Tubes and Caps Fragmentation of aRNA target before hybridization onto Affymetrix probe arrays has been shown to be critical in obtaining optimal assay sensitivity Affymetrix recommends that the aRNA used in the fragmentation procedure be sufficiently concentrated to maintain a small volume during the procedure This will minimize the amount of magnesium in the final hybridization cocktail Fragment an appropriate amount of aRNA for hybridization cocktail preparation and gel analysis aRNA amount depends on the format of the Affymetrix probe array you are using 1 Assemble the aRNA fragmentation mixture Table 3 2 Fragmentation Reaction Setup Component Amount per Array aRNA 10 ug 1 to 20 uL 5x Array Fragmentation Buffer 5 uL Nuclease free Water Variable up to 25 pL final volume Total Volume 25 pL Exact volume will depend on aRNA concentration 2 Fragmentation Reaction A Incubate the fragmentation reaction at 94 C for 35 m
16. cription to Synthesize Biotin Modified aRNA with IVT Labeling Master Mix generates multiple copies of biotin modified aRNA from the double stranded cDNA templates this is the amplification step n Optional stopping point The aRNA can be stored overnight at 20 C at this point 1f desired aRNA Purification removes unincorporated NTPs salts enzymes and inorganic phosphate to improve the stability of the biotin modified aRNA Fragmentation of the labeled aRNA prepares the target for hybridization to Affymetrix 3 expression array strips Contents 3 Figure 1 1 Overview of the GeneAtlas 3 IVT Express Kit Labeling Assay Approximate Total RNA Sample Time 58 BE EB 00810808100 I AAAAA S 1 Polv A RNA Control Pa Poly A RNA Controls Poly ontro PA T7 Oligo dT Primer 3 TTTTT MS 2 First strand i ax 235 ho rs cDNA Synthesis SPPPPPP PPP PAAAAA 3 300000 0 00 0 0 TTTTT ms 3 Second strand cDNA Synthesis v 1 5 hours S p IUHgllllll AAAAA mmm 3 LI D D Inmrnliriflrrrrr mms A INT Labeli ss Biotinylated P aug Ribonucleotide ours or Or ARNA v z Analog 16 hours SETTAT UUUUU 5 SELETATI I uuu 5 DR i SEELETLELELE EUUUUU 5 5 aRNA Purification Son 6 Fragmentation i 1 hour 7 Hybridization b 16 hours amp gt Legend TIIIL RNA TOooo DNA E T7 promoter Biotin 4 GeneAtlas 3 IVT Express Kit User Manual Important Parameters for Successful A
17. e for transcription n vitro transcription synthesizes aRNA and incorporates a biotin congugated nucleotide CRNA is also known as amplified RNA or aRNA The aRNA is then purified to remove unincorporated NTPs salts enzymes and inorganic phosphate Fragmentation of the biotin labeled aRNA prepares the sample for hybridization onto Affymetrix 3 expression array strips If you are using the GeneAtlas 3 IVT Express Kit for the first time it is recommended to use the control RNA to ensure that you get sufficient RNA yields for your array experiments Control RNA Use the included Control RNA to familiarize yourself with the GeneAtlas 3 IVT Express Kit RNA Amplification procedure Instructions for the positive control reaction are provided in Appendix A Troubleshooting on page 49 2 GeneAtlas 3 IVT Express Kit User Manual Assay Overview The GeneAtlas 3 IVT Express Kit aRNA amplification procedure is depicted in Figure 1 1 Reverse Transcription to Synthesize First Strand cDNA is primed with T7 oligo dT primer to synthesize cDNA containing a T7 promoter sequence Second Strand cDNA Synthesis converts the single stranded cDNA into a double stranded DNA dsDNA template for transcription The reaction employs DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA n Optional stopping point Samples can be stored overnight at 20 C at this point if desired In Vitro Trans
18. erials RNA Sample diluted to appropriate concentration Non stick RNase free Microfuge Tubes for Poly A Control RNA dilution See Note below x NOTE If frozen the Poly A Control Dil Buffer may take 15 to 20 minutes to thaw at room temperature Designed specifically to provide exogenous positive controls to monitor the entire eukaryotic target labeling process a set of poly A RNA controls is supplied in the GeneAtlas 3 IVT Express Kit Each eukaryotic Affymetrix probe array contains probe sets for several B subtilis genes that are absent in eukaryotic samples lys phe thr and dap These poly A RNA controls are in vitro synthesized and the polyadenylated transcripts for the B subtilis genes are premixed at staggered concentrations The concentrated Poly A Control Stock can be diluted with the Poly A Control Dil Buffer and spiked directly into RNA samples to achieve the final concentrations referred to as a ratio of copy number summarized below in Table 2 3 Table 2 3 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 Contents 15 The controls are then amplified and labeled together with the total RNA samples Examining the hybridization intensities of these controls on Affymetrix array strips helps to monitor the labeling process independe
19. gilent com Figure 1 2 Example Agilent Bioanalyzer Electropherograms from three different total RNAs of varying integrity Panel A represents a highly intact total RNA RIN 9 2 panel B represents a moderately intact total RNA RIN 6 2 and panel C represents a degraded total RNA sample RIN 3 2 FU FU FU A RIN 92 B RIN 6 2 120 25 200 S00 1000 2000 4500 nt 25 200 560 1000 2000 4000 nt 25 300 500 1000 2000 4000 nt NOTE Total RNAs with lower RIN values may require increased input amounts to generate enough aRNA for hybridization to an array Denaturing agarose gel electrophoresis and nucleic acid staining can also be used to separate and visualize the major rRNA species After purification RNA concentration is determined by absorbance at 260 nm on a spectrophotometer 1 absorbance unit 40 ug mL RNA The A 4 A ratio should be approximately 2 0 with ranges between 1 8 to 2 1 considered acceptable We recommend checking the quality of RNA by running it on an agarose gel prior to starting the assay When the RNA resolves into discrete rRNA bands 1 e no significant smearing below each band with the 28S rRNA band appearing approximately twice as intense as the 18S rRNA band then the mRNA in the sample is likely to be mostly full length The primary drawback to gel electrophoresis is that it requires microgram amounts of RNA 6 GeneAtlas 3 IVT Express Kit User Manual
20. given experiment 3 Place the aRNA on ice briefly or freeze immediately Place the reaction on ice and proceed to the aRNA purification step below or immediately freeze at 20 C for overnight storage EH TIP STOPPING POINT The aRNA can be stored overnight at 20 C at this point if desired 22 GeneAtlas 3 IVT Express Kit User Manual aRNA Purification Reagents and Materials Required Table 2 13 Reagents and Materials Required Item Needed GeneAtlas 3 IVT Express Kit Box 1 RNA Binding Beads aRNA Binding Buffer Concentrate aRNA Wash Solution aRNA Elution Solution U bottom Plate 8 Strip PCR Tubes and Caps for storage of purified aRNA User supplied Reagents and Materials 1 5 mL RNase free Tube for pre heating Elution Solution RNase free Microfuge Tubes for making master mix See Important note below bu IMPORTANT Make sure that the appropriate volume of ethanol has been added to the bottle of aRNA Wash Solution before use After synthesis the aRNA is purified to remove enzymes salts and unincorporated nucleotides Photos of the aRNA purification process can be found in Appendix B on page 51 If a plate shaker other than the recommended Lab Line Titer Plate Shaker will be used approximate shaking speeds for each step can be found in Appendix C Shaker Speeds on page 53 Before Beginning the aRNA Purification Preheat the aRNA Elu
21. he block temperature Otherwise incubate all reactions with the heated lid on 100 C Maintain procedural consistency Procedural consistency is very important for amplification experiments Consider implementing a detailed procedural plan that will be used by everyone in the lab to maintain consistency This type of plan will minimize variation due to subtle procedural differences that can influence RNA amplification and may complicate gene expression studies The plan should include basic information such as the method of RNA isolation the amount of RNA to use in the procedure and how long to incubate the IVT reaction It should also address specifics that are not often included in protocols such as which tubes and thermal cycler to use for each step in the process Finally develop a consistent work flow For example standardize stopping points in the method The idea is to standardize all of the variables discussed in this section of the manual and carefully follow all the protocol steps in order to maximize amplification consistency among samples Contents 7 Use Poly A RNA Controls to monitor the target labeling process The use of Poly A RNA Controls allows you to evaluate assay sensitivity consistency and dynamic range The kit contains four exogenous pre mixed poly adenylated prokaryotic controls that are spiked directly into RNA samples before target labeling Their resultant signal intensities on Affymetrix brand array stri
22. inutes B Place the reaction on ice immediately after the incubation 3 Optional Evaluate a sample of the reaction on a Bioanalyzer Analyze the size of the fragmentation reaction products by running a 300 ng sample of the reaction on an Agilent bioanalyzer using an Agilent RNA 6000 Nano Kit Figure 3 3 shows a typical result of such analysis Follow the manufacturer s instructions for this analysis The reaction should produce a distribution of 35 200 nt aRNA fragments with a peak at approximately 100 120 nt Contents 31 Figure 3 3 Example Agilent Bioanalyzer Electropherogram of fragmented aRNA FU i Fragmented aRNA 144 124 Peak Size 100 120 nt 25 200 500 1000 2000 4000 nt 4 Use fragmented aRNA immediately or store frozen Use the fragmented aRNA immediately or store undiluted fragmented aRNA at 20 C or 70 C for longer term storage 32 GeneAtlas 3 IVT Express Kit User Manual Hybridization This chapter outlines the basic steps involved in hybridizing your array strip s on the GeneAtlas System The three major steps involved in array strip hybridization are a GeneAtlas Software Setup on page 33 a Target Hybridization Setup for Affvmetrix Array Strips on page 38 a Hybridization of Array Strips on the GeneAtlas System on page 46 13 NOTE If you are using a hybridization ready sample or re hybridizing previously made hybridization cocktail start at the
23. ion cocktail needs to be denatured again prior to reapplication to a new array bus IMPORTANT Rehybridization of hybridization cocktails should only be necessary in case of serious array problems The performance of rehybridized samples has not been thoroughly tested and is recommended only when absolutely necessary Troubleshooting Positive Control Reaction Control RNA Amplification Instructions To verify that the process is working as expected a Control RNA sample isolated from HeLa cells is provided with the kit 1 Dilute 2 uL of the Control RNA into 18 uL of Nuclease free Water 2 Use luL of the diluted Control RNA 100 ng follow the protocol starting at Reverse Transcription to Synthesize First Strand cDNA on page 17 3 At In Vitro Transcription to Synthesize Labeled aRNA on page 20 use a 16 hour incubation for the IVT reaction 4 Continue with the procedure for making biotin modified aRNA through aRNA Purification on page 22 Expected Results The positive control reaction should produce gt 40 ug of aRNA a The average size of the aRNA should be 800 nucleotides Factors that Affect Both Positive Control and Experimental Samples If the positive control reaction yield or amplification product size does not meet expectations consider the following possible causes and troubleshooting suggestions These suggestions also apply to problems with amplification of experimental RNA Incubation Temperature s We
24. itation and fragmentation Purified aRNA can be stored at 20 C for up to 1 year As with any RNA preparation the number of freeze thaw cycles should be minimized to maintain aRNA integrity 26 GeneAtlas 3 IVT Express Kit User Manual Evaluation and Fragmentation of aRNA aRNA Quantitation and Expected Yield Assessing aRNA Yield by UV Absorbance The concentration of an aRNA solution can be determined by measuring its absorbance at 260 nm We recommend using NanoDrop Spectrophotometers for convenience No dilutions or cuvettes are needed just measure 2 uL of the aRNA sample directly Alternatively the aRNA concentration can be determined by diluting an aliquot of the preparation in TE 10 mM Tris HCl pH 8 1 mM EDTA and reading the absorbance in a traditional spectrophotometer at 260 nm Find the concentration in pg mL using the equation shown below 1 A 40 ug RNA mL Expected Yield The aRNA yield will depend on the amount and quality of poly A RNA in the input total RNA Since the proportion of poly A RNA in total RNA is affected by influences such as health of the organism and the organ from which it is isolated aRNA yield from equal amounts of total RNA may vary considerably Figure 3 1 shows yield data for aRNA produced with the kit from several different types of input RNA 28 GeneAtlas 3 IVT Express Kit User Manual Figure 3 1 Average aRNA Yield from a variety of total RNA samples RN
25. ization tray is not a sample well It is important that you do not add anything to this area Figure 4 8 Figure 4 8 Location of 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 Remove the array strip from its foil pouch and carefully place into the hybridization tray see Figure 4 9 making sure that there are no bubbles beneath the array strip CAUTION Be very careful not to scratch damage the array surface EH 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 Contents 41 Thin Thick Rift Thick C 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 D 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 E Place the array strip hybridization tray into the Hybridization Station at 45 C for 15 minutes ES WARNING Do not force the GeneAtlas Hybridization clamps up To open press down on the top of the clamp and simultaneously slightly lift the protr
26. ly A Control Dil Buffer to prepare the Second Dilution 1 50 Mix thoroughly and spin down to collect the liquid at the bottom of the tube 5 Add2 uL of the Second Dilution to 148 uL of Poly A Control Dil Buffer to prepare the Third Dilution 1 75 Mix thoroughly and spin down to collect the liquid at the bottom of the tube 7 Add2 uL of the Third Dilution to 18 uL of Poly A Control Dil Buffer to prepare the Fourth Dilution 1 10 16 GeneAtlas 3 IVT Express Kit User Manual 8 Mix thoroughly and spin down to collect the liquid at the bottom of the tube 9 Add 1 5 uL of this Fourth Dilution to 100 ng of total RNA at a concentration of 100 ng uL NOTE The first dilution of the poly A RNA controls can be stored up to six weeks in a non frost free freezer at 20 C and frozen thawed up to eight times Label the storage tube with the expiration date for future reference Table 2 5 Total RNA Poly A RNA Control Mixture Component Volume Total RNA Sample 50 500 ng 1 uL Diluted Poly A RNA Controls Fourth Dilution 1 5 uL Total Volume 2 5 pL Contents 17 Reverse Transcription to Synthesize First Strand cDNA Reagents and Materials Required Table 2 6 Reagents and Materials Required Item Needed GeneAtlas 3 IVT Express Kit Box 1 8 Strip PCR Tubes and Caps 0 2 mL GeneAtlas 3 IVT Express Kit Box 2 First Strand Buffer Mix First Strand Enzyme Mix Poly A Controls User
27. mplification Input RNA Quantity and IVT Reaction Incubation Time Consider both the type and amount of sample RNA available and the amount of aRNA needed for your analysis when planning experiments using the GeneAtlas 3 IVT Express Kit Because mRNA content varies significantly with tissue type the optimal amount of total RNA input and IVT incubation time should be determined empirically for each experimental system The recommended input RNA amounts listed in Table 1 1 are based on using total RNA from HeLa cells use these recommendations as a starting point Table 1 2 shows the corresponding recommended IVT incubation times Lus IMPORTANT Optimal RNA input amount and IVT incubation time are sample type dependent and should be determined empirically It is recommended to keep input amount and IVT incubation time consistent within a given experiment Table 1 1 Input RNA Limits Recommendations Amount Recommended 100 ng Minimum 50 ng Maximum 500 ng x NOTE The RNA needs to be diluted to an appropriate concentration such that the desired input amount is present in a 1 pL volume For example for 100 ng input RNA concentration should be 100 ng pL Table 1 2 Recommended IVT Incubation Times Recommendations RNA Amount IVT Incubation Time Recommended 50 250 ng 16 hours Optional 250 500 ng 4 hours RNA Purity RNA quality is the single most important factor affecting how efficiently an R
28. ntly from the quality of the starting RNA samples The Poly A RNA Control Stock and Poly A Control Dil Buffer are provided in the GeneAtlas IVT Express Kit to prepare the appropriate serial dilutions based on Table 2 4 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 Lu IMPORTANT Use non stick RNase free microfuge tubes to prepare all of the dilutions not included Table 2 4 Serial Dilution of Poly A RNA Control Stock Serial Dilutions Volume of 4th Input Amount Bree Dili dit Hd piluti h Diluti dilution to add irst Dilution Second Dilution ird Dilution Fourth Dilution to total RNA 1 10 1 50 1 75 1 20 1 5 uL 1 10 1 50 1 75 1 10 1 5 pL 1 10 1 50 1 75 1 4 1 5 uL 1 10 1 50 1 75 1 2 1 5 uL Recommendation Avoid pipetting solutions less than 2 uL in volume when making dilutions to maintain precision and consistency when preparing the dilutions For example to prepare the poly A RNA dilutions for 100 ng of total RNA 1 Add2 uL of the Poly A Control Stock to 18 uL of Poly A Control Dil Buffer for the first dilution 1 10 Mix thoroughly and spin down to collect the liquid at the bottom of the tube Add 2 uL of the First Dilution to 98 uL of Po
29. on a Add 100 ethanol ACS reagent grade or equivalent to the bottle labeled aRNA Wash Solution Concentrate The volume of ethanol required is listed on the bottle label of aRNA Wash Solution Concentrate Mix well and mark the label to indicate that the ethanol was added This solution will be referred to as aRNA Wash Solution in these instructions Store at room temperature Program the Thermal Cycler Incubate all reactions in a thermal cycler We find it convenient to set up the thermal cycler programs for each incubation before starting the procedure The specifications for each incubation are shown in Table 2 1 Table 2 1 Thermal Cycler Programs for RNA Amplification Program or Method First Strand cDNA Synthesis 42 C for 2 hrs 4 C indefinite hold Second Strand cDNA Synthesis 16 C for 1 hr 65 C for 10 min 4 C indefinite hold IVT 40 C for4 or 16 hrs 4 C indefinite hold Fragmentation 94 C for 35 min 4 C indefinite hold Pre Hybridization 96 C for 10 min 45 C for 2 min 45 C indefinite hold 14 GeneAtlas 3 IVT Express Kit User Manual Prepare Poly A RNA Controls Reagents and Materials Required Table 2 2 Reagents and Materials Required Item Needed GeneAtlas 3 IVT Express Kit Box 1 8 Strip PCR Tubes and Caps 0 2 mL GeneAtlas 3 IVT Express Kit Box 2 Poly A RNA Control Stock Poly A RNA Control Dil Buffer User supplied Reagents and Mat
30. on procedure impure RNA Samples RNA samples with significant amounts of contaminating DNA protein phenol ethanol or salts are reverse transcribed poorly and subsequently generate less aRNA than pure RNA samples Phenol extract and ethanol precipitate your RNA or use a commercially available RNA cleanup kit to further purify your RNA before reverse transcription RNA Integrity is Compromised RNA that is partially degraded generates cDNA that is relatively short This will reduce the average size of the aRNA population and subsequently reduce the yield of aRNA You can assess the integrity of an RNA sample by determining the size of the 18S and 28S rRNA bands and the relative abundance of 28S to 18S rRNA See RNA Integrity on page 5 for more information The mRNA Content of Your total RNA Sample is Lower Than Expected Different RNA samples contain different amounts of mRNA In healthy cells mRNA constitutes 1 10 of total cellular RNA Johnson 1974 Sambrook and Russell 2001 The actual amount of mRNA depends on the cell type and the physiological state of the sample When calculating the amount of amplification the starting mass of mRNA ina total RNA prep should always be considered within a range of 10 30 ng per ug of total RNA assuming good RNA quality aRNA Purification Photos Figure B 1 Photos of aRNA Purification Step figure 1 of 2 Ambion Magnetic Stand 96 Ambion 96 well Magnetic Ring Stand P N AM10027 P
31. p and down several times 3 aRNA binding A Add 60 uL 10096 ethanol to each sample B Mix by pipetting up and down several times 24 GeneAtlas 3 IVT Express Kit User Manual C Gently shake for 2 to 5 minutes to thoroughly mix setting 4 on the Lab Line Titer Plate Shaker The aRNA in the sample will bind to the RNA Binding Beads during this incubation Refer to Appendix C on page 53 for appropriate shaker speeds EH TIP Any unused wells should be covered with a plate sealer so that the plate can safely be reused 4 RNA Binding Beads capture A Move the plate to the magnetic stand and capture the magnetic beads for 5 minutes When capture is complete the mixture becomes transparent and the RNA Binding Beads will form aggregate against the magnets in the magnetic stand The exact capture time depends on the magnetic stand used and the amount of aRNA in your sample f NOTE For maximum aRNA recovery mix well and ensure that the mixture is transparent all of the beads have been captured before proceeding B Carefully remove and discard the supernatant without disturbing the magnetic beads C Remove the plate from the magnetic stand 5 Bead Washing IMPORTANT Make sure that ethanol has been added to the bottle of aRNA Wash Solution Concentrate before using it A Add 80 uL aRNA Wash Solution to each sample and shake at moderate speed for minute setting 7 on the Lab Line Titer Plate Shake
32. paration for Second Strand cDNA Synthesis B After the incubation centrifuge briefly 5 seconds to collect the first strand cDNA at the bottom of the tube plate Place the sample on ice and immediately proceed to Second Strand cDNA Synthesis on page 18 Second Strand cDNA Synthesis Reagents and Materials Required Table 2 8 Reagents and Materials Required Item Needed GeneAtlas 3 IVT Express Kit Box 2 Nuclease free Water Second Strand Buffer Mix Second Strand Enzyme Mix User supplied Reagents and Materials RNase free Microfuge Tubes for making master mix 1 Pre cool thermal cycler block to 16 C 2 Assembly of Second Strand Master Mix A On ice prepare a Second Strand Master Mix in a nuclease free tube in the order listed in Table 2 9 Prepare master mix for all the samples in the experiment including 5 overage to cover pipetting error Table 2 9 Second Strand Master Mix Component Amount Used for Master Mix for 1 Sample 4 Samples Includes 596 Overage Nuclease free Water 6 5 uL 27 3 uL Second Strand Buffer Mix 2 5 uL 10 5 uL Second Strand Enzyme Mix 1 uL 4 2 uL Total Volume 10 pL 42 0 uL Contents 19 Mix well by gently vortexing Centrifuge briefly 5 seconds to collect the mix at the bottom of the tube and place on ice Transfer 10 uL Second Strand Master Mix to each 5 uL cDNA sample Mix thoroughly by gently vortexing or flicking the tube 3
33. plies Table 1 7 Lab Equipment and Supplies Contents 11 Material Source P N Lab Equipment and Supplies Thermal Cycler with heated Lid with appropriate adaptersto multiple accommodate strip tubes capable of holding 0 2 mL tubes for reaction incubations Vortex Mixer with flat top adapter for strip tubes multiple Microcentrifuge with an adapter for the PCR strip tubes or multiple plates supplied with the kit Magnetic Stand for 96 well plates Ambion AM10050 96 well Magnetic Stand or AM10027 Magnetic Stand 96 Orbital shaker for 96 well plates multiple e g Barnstead Lab Line Titer Plate Shaker Vacuum Centrifuge Concentrator Optional Spectrophotometer NanoDrop ND 8000 e g NanoDrop ND 8000 UV Vis Spectrophotometer Technologies Reagents and apparatus for preparation and electrophoresis of agarose gels Optional Miscellaneous Supplies Pipette for 0 1 to 2 uL Rainin L 2 Pipette for 2 to 20 uL Rainin L 20 Pipette for 20 to 200 uL Rainin L 200 Pipette for 100 to 1000 pL Rainin L 1000 Sterile barrier RNase free Pipette Tips multiple Bioanalyzer Agilent Non stick RNase free microfuge tubes 0 5 mL Ambion N12350 Non stick RNase free microfuge tubes 1 5 mL Ambion 12450 Or equivalent 12 GeneAtlas 3 IVT Express Kit User Manual aRNA Amplification Protocol Equipment and Reagent Preparation Prepare aRNA Wash Soluti
34. ps serve as sensitive indicators of the labeling reaction efficiency independent from starting sample quality 8 GeneAtlas 3 IVT Express Kit User Manual Kit Contents and Storage Conditions Table 1 3 GeneAtlas 3 IVT Express Kit Components and Storage Conditions Component Vol Qnty 20 Rxn Storage BOX 1 of 2 aRNA Binding Buffer Concentrate 600 uL room temp RNA Binding Beads 120 uL 2 8 C aRNA Wash Solution Concentrate Add 8 mL 100 10 mL room temp ethanol before use as shown on the label aRNA Elution Solution 5 mL room temp Nuclease free Water 10 mL room temp 5X Array Fragmentation Buffer 1 mL room temp 8 Strip PCR Tubes amp Caps 0 2 mL 10 ea room temp U Bottom Plate 1 ea room temp Reservoir 1 ea room temp BOX 2 of 2 First Strand Enzyme Mix 11 uL 20 C First Strand Buffer Mix 44 uL 20 C Second Strand Enzyme Mix 22 uL 20 C Second Strand Buffer Mix 55 uL 20 C IVT Enzyme Mix 66 uL 20 C IVT Labeling Buffer 220 uL 20 C IVT Biotin Label 44 uL 20 C Control RNA 1 mg mL HeLa total RNA 10 uL 20 C Nuclease free Water 1 75 mL 20 C Poly A Control Stock 16 uL 20 C Poly A Control Dilution Buffer 3 8 mL 20 C 20X Hybridization Controls 450 uL 20 C Control Oligo B2 150 uL 20 C Do not freeze Contents Table 1 4 GeneAtlas Hybridization Wash and Stain Kit for 3 IVT Arrays P N 901531 9
35. r NOTE The RNA Binding Beads may not fully disperse during this step this is expected and will not affect RNA purity or yield B Move the plate to a magnetic stand and capture the RNA Binding Beads for 5 minutes as in the previous step Beads may have a clumpy appearance Please refer to Appendix B aRNA Purification Photos on page 51 C Carefully aspirate and discard the supernatant without disturbing the RNA Binding Beads and remove the plate from the magnetic stand D Repeat Step A through Step C to wash a second time with 80 uL of aRNA Wash Solution E Move the plate to a shaker and shake the plate dry vigorously for 1 minute to evaporate residual ethanol from the beads setting 10 on the Lab Line Titer Plate Shaker Refer to Appendix C on page 53 for appropriate shaker speeds 6 Contents 25 aRNA Elution A Elute the purified aRNA from the RNA Binding Beads by adding 30 uL preheated 50 60 C aRNA Elution Solution to each sample B Vigorously shake the plate for 3 minutes setting 10 on the Lab Line Titer Plate Shaker Then check to make sure the RNA Binding Beads are fully dispersed If they are not continue shaking until the beads are dispersed C Move the plate to a magnetic stand and capture the RNA Binding Beads for approximately 5 minutes D Transfer the supernatant which contains the eluted aRNA to a nuclease free PCR tube Store aRNA at lt 20 C or place on ice and proceed with quant
36. re Incorrect The incubation temperatures are critical for effective RNA amplification Use only properly calibrated thermal cyclers for the procedure Condensation formed in the tube during the reaction incubation s Condensation occurs when the cap of the reaction vessel is cooler e g room temperature than the bottom of the tube As little as 1 2 uL of condensate in an IVT reaction tube throws off the concentrations of the nucleotides and magnesium which are crucial for good yield If you see condensation check to make sure that the heated lid feature of the thermal cycler is working properly 50 GeneAtlas 3 IVT Express Kit User Manual Nuclease Contamination Using pipettes tubes or equipment that are contaminated with nucleases can cleave the RNA or DNA being generated at each step in the procedure This will reduce the size of the aRNA products and decrease aRNA yield Both RNases and DNases can be removed from surfaces using a RNase decontamination solution such as RNaseZap Troubleshooting Low Yield and Small Average aRNA Size Consider the following troubleshooting suggestions if the positive control reaction produced the expected results but amplification of your experimental samples results in less or smaller average lt 500 nt aRNA than expected Lower Than Expected Input RNA Concentration Take another A260 reading of your RNA sample or if it is available try using 100 250 ng of RNA in the amplificati
37. ridization tray containing the pre hybridization buffer and place it into the hybridization tray containing the hybridization cocktail samples D Check for and remove any bubbles that were introduced Refer to Figure 4 12 for proper orientation of the array strip in the hybridization tray IMPORTANT Insertion of the array strip and air bubble removal should be performed quickly to avoid drying of the array surface 44 GeneAtlas 3 IVT Express Kit User Manual Thin Thick Rift Thick E Place the hybridization tray with the array strip into a clamp inside the Hybridization Station and close the clamp as shown in Figure 4 13 Contents 45 6 Proceed to Hybridization of Array Strips on the GeneAtlas System on page 46 46 GeneAtlas 3 IVT Express Kit User Manual Hybridization of Array Strips on the GeneAtlas System After the array strip are registered in the GeneAtlas Software and the target hybridization steps are complete processing on the GeneAtlas System may begin 1 Return to the GeneAtlas Software interface Figure 4 14 should be visible Add Strip Scan or enter the bar code Bar Code Strip Name Time 30 Temperature 48 C aco 2 With the hybridization tray and array strip already in the GeneAtlas Hybridization Station click Start in Figure 4 14 The software displays the hybridization time countdown This time is displayed with a
38. ster Mix in a nuclease free tube in the order listed in Table 2 11 Prepare master mix for all the samples in the experiment including 5 overage to cover pipetting error Table 2 11 IVT Master Mix Component Amount Used for Master Mix for 1 Sample 4 Samples Includes 5 Overage IVT Biotin Label 2 uL 8 4 uL IVT Labeling Buffer 10 uL 42 0 uL IVT Enzyme Mix 3 uL 12 6 uL Total Volume 15 pL 63 0 uL B Mix well by gently vortexing Centrifuge briefly 5 seconds to collect the mix at the bottom of the tube and place on ice C Transfer 15 uL of IVT Master Mix to each 15 uL double stranded cDNA sample Mix thoroughly by gently vortexing and centrifuge briefly to collect the reaction at the bottom of the tube plate D Once assembled place the reaction in the thermal cycler block 2 Incubation Contents 21 Incubate the IVT reaction for 4 or 16 hours at 40 C in a thermal cycler using the program for IVT Table 2 1 on page 13 The recommended incubation time is based on the amount of input RNA and is shown in Table 2 12 Table 2 12 Recommended IVT Incubation Times Recommendations RNA Amount IVT Incubation Time Recommended 50 250 ng 16 hours Optional 250 500 ng 4 hours bu IMPORTANT Optimal RNA input amount and IVT incubation time are sample type dependent and should be determined empirically It is recommended to keep input amount and IVT incubation time consistent within a
39. ted Results 49 Factors that Affect Both Positive Control and Experimental Samples 49 Troubleshooting Low Yield and Small Average aRNA Size 50 aRNA Purification Photos x 225 were d m RE Rer dr 51 Shaker Speeds MOM 53 Overview The GeneAtlas 3 IVT Express Kit is the latest technology in RNA target preparation for microarray expression analysis This kit features Low RNA input requirements from as little as 50 ng of total RNA for a single round of amplification Streamlined workflow with the option to decrease target labeling time to a single day with appropriate inputs of total RNA Master mixes consumables included and a simple protocol for ease of use convenience and a high rate of success A complete kit that includes Poly A RNA controls and hybridization controls Magnetic bead aRNA purification for high recovery and ease of use The kit is based upon linear RNA amplification and employs T7 in vitro transcription technology Also known as the Eberwine or reverse transcription IVT RT IVT method this process 1s considered the gold standard for target preparation for gene expression analysis RT IVT was experimentally validated using TaqMan RT PCR MAQC Consortium et al 2006 In the GeneAtlas 3 IVT Express Protocol total RNA undergoes reverse transcription to synthesize first strand cDNA This cDNA is then converted into a double stranded DNA templat
40. tide B2 3 nM 2 5 uL 11 uL 50 pM 20X Hybridization Controls 7 5 uL 33 uL 1 5 5 25 and bioB bioC bioD cre 100 pM respectively 1 3X Hybridization Solution A 40 uL 176 uL 1X 1 3X Hybridization Solution B 75 uL 330 uL 1X Nuclease free Water 6 2 uL 27 3 uL Total Master Mix Volume 131 2 pL 577 3 uL Fragmented and Labeled aRNA 18 8 uL 7 5 ug 0 05 ug uL Total Volume 150 pL See Important note on page 39 IMPORTANT It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Controls are heated to 65 C for 5 minutes to completely resuspend the aRNA before aliquoting B Add 131 2 uL of the hybridization cocktail Master Mix to 7 5 ug 18 8 uL of fragmented and labeled aRNA C Mix thoroughly by gently vortexing Centrifuge briefly to collect hybridization mix at the bottom of the tube Place tube on ice or store at 20 C for longer term storage 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 Refer to Rehybridizing Used Cocktails on page 48 for additional information 40 GeneAtlas 3 IVT Express Kit User Manual 3 Pre Hybridization of the Array Strip A Gently pipette 120 uL of Pre Hybridization Buffer into the appropriate wells of the hybridization tray see Figure 4 8 Avoid generating air bubbles PA CAUTION The center of the hybrid
41. tion Solution to 60 C for at least 10 minutes x NOTE Aliquot the appropriate amount of aRNA Elution Solution 30 pL per sample plus 10 overage to a separate 1 5 mL RNase Free Tube not included to insure thorough pre heating of the Elution Solution Contents 23 Table 2 14 Preparation of aRNA Elution Solution Component Amount Used for Amount for 1 Sample 4 Samples Includes 10 Overage aRNA Elution Solution 30 uL 132 uL 1 Preparation of aRNA Binding Mix NOTE This entire procedure is performed at room temperature E IMPORTANT Prepare only the amount needed for all samples in the experiment plus 10 overage to cover pipetting error At room temperature assemble aRNA Binding Mix in a nuclease free tube for all the samples in the experiment following the instructions in Table 2 15 Table 2 15 aRNA Binding Mix Preparation Instructions for a single reaction Component Amount Used for Master Mix for 1 Sample 4 Samples Includes 10 Overage RNA Binding Beads 5 uL 22 ul aRNA Binding Buffer Concentrate 25 uL 110 uL Total Volume 30 pL 132 uL Do not put beads on ice Ensure that the beads are fully suspended by gently flicking the tube f there is a precipitate present vortex until precipitate dissipates 2 Addition of aRNA Binding Mix A Add 30 uL aRNA Binding Mix to each sample B Transfer each sample to a well of a U Bottom Plate C Mix by pipetting u
42. uding lever to unlock The clamp should open effortlessly Refer to Figure 4 10 42 GeneAtlas 3 IVT Express Kit User Manual Figure 4 10 Opening the clamps on the GeneAtlas Hybridization Station 1 Push Down 2 Lift Lever im ud Ini 4 Array Strip Sample Preparation To be carried out during array strip pre hybridization A For each sample to by hybridized pipette 120 uL of the hybridization cocktail from Step 2 into a 0 2 mL PCR Strip Tube B Incubate at 96 C for 10 minutes in a thermal cycler followed by at least 2 minutes at 45 C using the program for Pre Hybridization Table 2 1 on page 13 5 Array Strip Sample Hybridization A After 10 15 minutes of pre hybridization remove the array strip from the Hybridization Station and place on bench top keeping the arrays immersed in the pre hybridization solution B Apply the 120 uL of pre heated hybridization cocktail to the middle of the appropriate wells of a new clean hybridization tray see Figure 4 11 IMPORTANT Do not add more than 120 pL of hybridization cocktail to the wells as that could result in cross contamination of the samples Contents 43 Figure 4 11 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 C Carefully remove the array strip from the hyb
43. white background Figure 4 15 When the countdown has completed the display turns yellow and the time begins to count up HYBRIDIZATION Hyb001 00 00 27 45 C Contents 47 KS Affymetrix HYBRIDIZATION Hyb001 45 C 3 When hybridization has completed click the Stop button in the upper right corner A confirmation message box appears Figure 4 17 A Are you sure you want to stop un e 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 refer to Rehybridizing Used Cocktails on page 48 for additional information Immediately proceed to the GeneAtlas Wash Stain and Scan protocol Please refer to GeneAtlas System User s Guide P N 08 0246 for further detail 48 GeneAtlas 3 IVT Express Kit User Manual Rehybridizing Used Cocktails A used hybridization cocktail can be rehybridized to a new array if necessary Collect the used hybridization cocktail immediately after the Fluidics run is completed add to the remainder of the hybridization cocktail master mix from Step 2D on page 39 and store at 20 C For rehybridization continue the protocol from Step 3 on page 40 The hybridizat
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