Ambion® WT Expression Kit - Thermo Fisher Scientific
Contents
1. Component 10 Reaction 30 Reaction 24 Reaction 96 Reaction Storage kit kit kit kit Part no Part no Part no Part no 4411973 4411974 4440537 4440536 First Strand Enzyme Mix 11 uL 33 uL 50 uL 150 uL 20 C First Strand Buffer Mix 44 uL 132 uL 160 uL 600 uL 20 C Second Strand Enzyme Mix 55 uL 165 uL 175 uL 650 uL 20 C Second Strand Buffer Mix 138 uL 413 uL 400 uL 1 45 mL 20 C IVT Enzyme Mix 66 uL 198 uL 210 uL 850 uL 20 C IVT Buffer Mix 264 uL 792 uL 750 uL 1 40 mL 20 C Control RNA 1 mg mL HeLa total RNA 10 uL 10 uL 10 uL 10 uL 20 C Nuclease free Water 1 75 mL 1 75 mL NA NA any temp 2nd Cycle Buffer Mix 88 uL 264 uL 425 uL 1 45 mL 20 C Random Primers 22 uL 66 uL 100 uL 300 uL 20 C 2nd Cycle Enzyme Mix 88 uL 264 uL 275 uL 1 00 mL 20 C RNase H 22 uL 66 uL 100 uL 300 uL 20 C Nucleic Acid Purification Reagents Nucleic Acid Binding Buffer Concentrate 1 1 mL 3 3 mL 3 5 mL 12 5 mL room temp Nucleic Acid Binding Beads 220 uL 660 uL 700 uL 2 5 mL 4 C Nucleic Acid Wash Solution Concentrate 10 mL 20 mL 15 mL 50 mL room temp Elution Solution 5 mL 5 mL 6 mL 22 mL 49C or room temp Nuclease free Water 10 mL 10 mL NA NA room temp Consumables 8 Strip PCR Tubes amp Caps 0 2 mL 10 40 NA NA room temp U Bottom Plate 2 4 NA NA room temp Reservoir 1 2 NA NA room temp t Store the Nuclease free Water at 20 C 4 C or room temp i Do not freeze Ambion9 WT Expression Kit User Guid2. Place the reaction on ice then proceed immediately to Fragment and label the single stranded cDNA on page 28 or freeze the samples at 20 C for overnight storage STOPPING POINT Samples can be stored overnight at 20 C Expected cDNA Yield During development of this kit using a wide variety of tissue types 10 ug of input cRNA yielded 6 to 9 ug of cDNA For most tissue types the recommended 10 ug of input cRNA should yield 6 ug of cDNA 1 Determine cDNA yield by UV absorbance Determine the concentration of a cDNA solution by measuring its absorbance at 260 nm We recommend evaluating the absorbance of 1 5 uL of cDNA sample using a NanoDrop Spectrophotometer Alternatively determine the cDNA concentration 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 Calculate the concentration in ug mL using the equation below 1 A565 33 ug DNA mL A goxdilution factorx33 ug DNA mL Note The equation above applies only to single stranded cDNA Optional Use Quant iT PicoGreen RNA Reagent to Assess cRNA yield If a fluorometer or a fluorescence microplate reader is available use the Quant iT PicoGreen RNA Reagent Invitrogen to measure RNA concentration Follow the manufacturer s instructions Optional Expected cDNA size distribution The expected cDNA profile does not resemble the cRNA profile The
3. WT Expression Kit r Procedure b Immediately after the incubation centrifuge briefly 5 sec to collect the cDNA at the bottom of the tube plate Place the sample on ice and immediately proceed to Hydrolyze using RNase H on page 25 Hydrolyze using In this procedure RNase H degrades the cRNA template leaving single stranded RNase H cDNA 1 Add 2 uL of RNase H to the 2nd Cycle cDNA Note For this section review the safety information on page 31 a On ice add 2 uL of RNase H to the 2nd Cycle cDNA from above Mix by pipetting up down 3 times to ensure that all of the enzyme is dispensed from the pipet tip b Mix thoroughly by gently vortexing Centrifuge briefly 5 sec to collect the reaction at the bottom of the tube plate Proceed immediately to the next step 2 Incubate for 45 min at 37 C then 5 min at 95 C then for at least 2 min at 4 C a Incubate in a thermal cycler using the RNase H Hydrolysis method for thermal cycling that is shown in Table 1 on page 14 IMPORTANT Cover the reactions with the heated lid at 75 C b After the incubation centrifuge briefly 5 sec to collect the RNase H Hydrolyzed 2nd Cycle cDNA at the bottom of the tube plate Place the samples on ice then proceed immediately to the next section Purify 2nd cycle cDNA STOPPING POINT Samples can be stored overnight at 20 C Pu rify 2nd cyc le After synthesis the second strand cDNA is purified to remove enzymes s
4. After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply WARNING HAZARDOUS WASTE from instruments Waste produced by the instrument is potentially hazardous Follow the guidelines noted in the preceding General Chemical Handling warning AN WARNING 4L Reagent and Waste Bottle Safety Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position 32 Ambion WT Expression Kit User Guide Appendix Safety Biological hazard safety Biological hazard safety Q WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provi
5. Documentation and Support section in this document Ambion WT Expression Kit User Guide 31 Appendix Safety Chemical safety Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage
6. arrays helps to monitor the labeling process independently from the quality of the starting RNA samples The Poly A RNA Control Stock and Poly A Control Dil Buffer are provided in the Affymetrix GeneChip Poly A RNA Control Kit Part no 900433 or equivalent to prepare the appropriate serial dilutions based on Table 3 This is a guideline when 50 100 250 or 500 ng of total RNA is used as starting material For starting sample amounts other than those listed here calculations are needed in order to perform the appropriate dilutions to arrive at the same proportionate final concentration of the spike in controls in the samples Table 3 Serial dilution of Poly A RNA Control Stock Serial dilutions Total RNA input Biss ef el amount 1st 2nd 3rd 4th AMAN to 200 te dilution dilution dilution dilution total RNA 50 ng 1 20 1 50 1 50 1 20 2 uL 100 ng 1 20 1 50 1 50 1 10 2uL 250 ng 1 20 1 50 1 50 TA 2uL 500 ng 1 20 1 50 1 50 1 2 2 uL Recommendation Avoid pipetting solutions less than 2 uL in volume to maintain precision and consistency when preparing the dilutions IMPORTANT Use non stick RNase free microfuge tubes to prepare all of the dilutions not included For example to prepare the Poly A RNA dilutions for 100 ng of total RNA 1 Add 2 uL of the Poly A Control Stock to 38 uL of Poly A Control Dil Buffer for the first dilution 1 20 2 Mix thoroughly and spin down to collect th
7. distribution A240 290 A290 Ratio Frequency nm Fragment and label the single stranded cDNA For instructions on how to fragment the single cDNA stranded cDNA refer to the Affymetrix GeneChip i WT Terminal Labeling and Hybridization User Manual Part no 702880 e UDG e APE1 TdT DLR Label 12 Ambion WT Expression Kit User Guide Procedure Implement a plan to maintain procedural consistency Equipment and reagent preparation Ambion WT Expression Kit IN Procedure To minimize sample to sample variation that is caused by subtle procedural differences in gene expression assays consider implementing a detailed procedural plan The plan standardizes the variables in the procedure and should include the Method of RNA isolation e Amount of input RNA that is used for each tissue type Type of thermal cycler that is used e Workflow stopping points Recommended thermal cycler Life Technologies recommends using the Applied Biosystems Veriti 96 Well Thermal Cycler with 0 2 mL sample wells Part no 4375786 to prepare samples using the Ambion WT Expression Kit If you use a different instrument make sure that the heated cover of your thermal cycler either tracks the temperature of the thermal cycling block or supports specific temperature programming Program the thermal cycler When using the Applied Biosystems Veriti Thermal Cycler set the idle temperature for the heated
8. lid to 50 C prior to starting Day 1 first strand cDNA synthesis Maintain this setting throughout Day 1 Prior to starting Day 2 2nd cycle cRNA denaturation change the idle temperature for the lid to 75 C Maintain this setting throughout Day 2 Prior to starting cDNA fragmentation change the idle temperature for the lid to 105 C which ensures that the temperature of the heated lid is at or near the required temperature for each step For instructions on how to change the temperature settings of the Veriti thermal cycler refer to the Applied Biosystems Veriti Thermal Cycler User Guide Part no 4375799 An alternate protocol may be used for thermal cyclers that lack a programmable heated lid This is not the preferred method Yields of cRNA may be greatly reduced if a lid heated to 105 C is used during the Second Strand cDNA Synthesis or during the In Vitro Transcription cRNA Synthesis steps We recommend leaving the heated lid open during Second Strand cDNA Synthesis A small amount of condensation will form during the incubation This is expected and should not significantly decrease cRNA yields For In Vitro Transcription cRNA Synthesis we recommend incubating the reaction in a 40 C hybridization oven if a programmable heated lid thermal cycler is unavailable Incubation temperatures and times are critical for effective RNA amplification Use properly calibrated thermal cyclers and adhere closely to the incubation times Ambion WT Expr
9. median cDNA size is approximately 400 nt This step is optional 1 Optional Determine cDNA size distribution using a Bioanalyzer Instrument Ambion WT Expression Kit User Guide 27 Ambion WT Expression Kit Troubleshooting We recommend analyzing cDNA size distribution using an Agilent 2100 Bioanalyzer a RNA 6000 Nano Kit Part no 5067 1511 and mRNA Nano Series II assay If there is sufficient yield load approximately 250 ng of cDNA per well If there is not sufficient yield then use as little as 200 ng of cDNA per well To analyze cDNA size using a Bioanalyzer Instrument follow the manufacturer s instructions Fragment and label the single stranded cDNA The Affymetrix GeneChip9 WT Terminal Labeling Kit Part no 900671 or equivalent is used for the fragmentation and labeling of the cDNA Because the 2nd cycle sense strand cDNA contains dUTP the kit uses uracil DNA glycosylase UDG and apurinic apyrimidinic endonuclease 1 APE1 to recognize and fragment the cDNA at the unnatural dUTP residues DNA is then labeled by terminal deoxynucleotidyl transferase TdT using the Affymetrix proprietary DNA Labeling Reagent For instructions on how to fragment and label the single stranded cDNA refer to the Affymetrix GeneChip WT Terminal Labeling and Hybridization User Manual Part no 702880 Troubleshooting Observation Possible cause Solution The positive control sample and your total RNA sa
10. Affymetrix Inc 2011 Life Technologies Corporation All rights reserved Part Number 4425209 Rev D August 2011 Contents About This Guide cupra ae ak bl y Park ee ay ee USE CE 5 Purpose a xax daya ete Qule ka gla kw k Rut ead guint eet A e 200A QED 2 een Dn tn 5 User attention WOndS uuu dikan n ceti pada et MIK KA T T ag eet Det Qira ERR 5 Ambion WT Expression Kit seen nnn 7 Produet information s a s55 mee nt ku Ke E tae AN d kala K Kin e See ees Yee 7 Purpose of the product g3 i ss cece eae a eee ehh 7 Kit contents and Storage 2sci044 2 220e 4 Sa ewl nk kuna dal cena me ceili aeons 8 Required materials vk kk kk kk kk kk kk kK kk kK kk kK kK kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk lk 9 Equlprment i iet ib ili A W N ANE SAN din id nb rene tcd WAYA 9 Reagents and suppliers yak Say er Copertina 9 Day T workflow cssc Ld el Rep EUR eU Re RO EN Vue ID A eee dp EET ug 10 Day 2 WOEKTIOW suec ce ce eit tee ewe taper ep ad C eas 11 Day 2 workflow continued WWW W kk kk kk kk kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk rn 12 Proced re essene via eian Aa E E mm 13 Implement a plan to maintain procedural consistency WWW kk kk kk kK KK KK KE KK 13 Equipment and reagent preparation WWW kk kk kk kk kk kK kK kK KK KK KK KK KK KK KK eee eee 13 Prepare the Control RNA and total RNA WWW kk kk kk kk kk kk KK KK KK KK KK KK KK KK KK KK KIR 14 Prepare the Poly A R
11. Binding Beads and discard the supernatant a Move the plate to a magnetic stand to capture the magnetic beads When capture is complete after 5 min the mixture is transparent and the Nucleic Acid Binding Beads form pellets against the magnets in the magnetic stand The exact capture time depends on the magnetic stand that you use and the amount of cRNA generated by in vitro transcription b Carefully aspirate and discard the supernatant without disturbing the magnetic beads then remove the plate from the magnetic stand 5 Wash twice with 100 uL of Nucleic Acid Wash Solution IMPORTANT Make sure that 100 ethanol has been added to the bottle of Nucleic Acid Wash Solution Concentrate before using it Ambion WT Expression Kit User Guide 21 Ambion WT Expression Kit Procedure a Add 100 uL of Nucleic Acid Wash Solution to each sample then shake at moderate speed for 1 min setting 7 on the Lab Line Titer Plate Shaker Note Although the Nucleic Acid Binding Beads may not fully disperse during this step this does not affect RNA purity or yield b Move the plate to a magnetic stand and capture the Nucleic Acid Binding Beads as in the previous step c Carefully aspirate and discard the supernatant without disturbing the Nucleic Acid Binding Beads then remove the plate from the magnetic stand d Repeat step 5a to step 5c to wash each sample again with 100 uL of Nucleic Acid Wash Solution e Move the plate t
12. NA RNA Controls Control Kit Part no 900433 or equivalent add the reagents to the total RNA samples Follow the instructions below We strongly recommend the use of poly A controls for all reactions that will be hybridized to GeneChip arrays Designed specifically to provide exogenous positive controls to monitor the entire target labeling process a set of poly A RNA controls supplied by Affymetrix can be added to the RNA prior to the First Strand Synthesis step Ambion WT Expression Kit User Guide 15 16 Ambion WT Expression Kit Procedure Each eukaryotic GeneChip probe array contains probe sets for several B subtilis genes that are absent in eukaryotic samples lys phe thr and dap These poly A RNA controls are in vitro synthesized and the polyadenylated transcripts for the B subtilis genes are premixed at staggered concentrations The concentrated Poly A Control Stock can be diluted with the Poly A Control Dil Buffer and spiked directly into RNA samples to achieve the final concentrations referred to as a ratio of copy number summarized in Table 2 Table 2 Final concentrations of Poly A RNA Controls when added to total RNA samples Poly A RNA Spike Final concentration ratio of copy number lys 1 100 000 phe 1 50 000 thr 1 25 000 dap 1 6 667 The controls are then amplified and labeled together with the total RNA samples Examining the hybridization intensities of these controls on GeneChip
13. NA Controls n nananana nannaa nnen kk kk kk kK kk kk kk kk kk kk kk lk 15 Evaluate RNA quality Wl ka kk kk kk kk kk kk kk kK kK kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk 17 Evaluate RNA integrity isse pex b LEER Le Leste pdt eee 17 Synthesize first strand cDNA WW kk kk kk kk kk kK kk KK KK KK KK KK len 17 Synthesize second strand cDNA WW kk kk kk kk kk kk kk kK kK kk kK kk kk kk kk kk kk kk kk k 19 Synthesize cRNA by In Vitro Transcription WW kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK 20 uri c nA TAE TEE rk ei E Rd 20 Assess CRNA yield and size distribution kk kk kk kk kk kK kK KK KK KK KK KK KK KK KK KK KK 22 Synthesize 2nd cycle cDNA kk kk kk kk kk kk kk kk kk kk kk kk kk kk e 23 Hydrolyze using RNase HW vk kk kk kk kk kk kk kk kk kk kk kK kk kk kk kk kK kk kk kk kk kk kk kk kk kk 25 Purify 2nd cycle cDNA A3 akuya deha nake ER AR ERA PAPA eed ERN ne 25 Assess cDNA yield and size distribution kk kk kk kk kK kK KK KK KK KK KK KK KK KK KK KK KK 27 Fragment and label the single stranded cDNA 2200000 cece cece KRKKK KK 28 Troubleshooting crecer ned ya ark he dde He Ged denne Heed dene Re ee AE 28 Ambion WT Expression Kit User Guide 3 Contents APPENDIX Safety em anre duae wares dom des marae pia acs 31 Chemicalsatety ctn dois c tini Poh ee viral er te ir aet dote do a tes 32 Biological hazard safety M K kk kk kk kk kK kk kk KK KK KK KK kK kK KK kk kk kk kK kk kk kk kk kk kk ees 33 Biblig ra
14. USER GUIDE 7 o ambion oy fe technologies Ambion WT Expression Kit for use with Affymetrix GeneChip Whole Transcript WT Expression Arrays Publication Part Number 4425209 Rev D Revision Date August 2011 technologies For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER LIMITED LICENSE Limited Use Label License Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal researc
15. action Note The positive control reaction should produce gt 20 ug of cRNA and gt 6 ug of 2nd cycle cDNA Prepare your total RNA Prepare your total RNA according to your laboratory s procedure Ambion9 WT Expression Kit User Guide Procedure Ambion WT Expression Kit Determine your input RNA quantity Consider both the type and amount of sample RNA that are available when planning your experiment Because mRNA content varies significantly with tissue type determine the total RNA input empirically for each tissue type or experimental condition The recommended total RNA inputs in the table below are based on total RNA from HeLa cells Use these values as reference points for determining your optimal RNA input RNA input criterion Total RNA ng Recommended 100 Minimum 50 Maximum 500 Figure 1 shows the cRNA yields with varying inputs of three RNAs the HeLa Kit control Stratagene Universal Human Reference RNA UHRR and Ambion FirstChoice Human Brain Reference RNA HBRR For each RNA the plot displays the yields from eight titration points 0 ng 12 5 ng 25 ng 50 ng 100 ng 200 ng 400 ng and 800 ng Figure 1 cRNA yields with varying inputs of three RNAs 350 o RNA Type UHRR HBRR HeLa Yield ug T T T T T T T T 100 0 100 200 300 400 500 600 700 800 900 Input ng Prepare the Poly A Note To include premixed controls from the Affymetrix GeneChip Poly A R
16. althcare Bio Sciences Corp s technology or intellectual property other than expressly provided herein End Users may not attempt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services DISCLAIMER OF WARRANTIES GE HEALTHCARE BIO SCIENCES CORP PROVIDES NO WARRANTIES TO END USER STATUTORY OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO PRODUCT QUALITY CONDITION DESCRIPTION MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND ALL SUCH WARRANTIES ARE HEREBY EXPRESSLY DISCLAIMED GE HEAL THCARE BIO SCIENCES CORP HEREBY EXPRESSLY DISCLAIMS ANY WARRANTY REGARDING RESULTS OBTAINED THROUGH THE USE OF THE PRODUCTS INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE INVALID OR INCOMPLETE RESULTS Exclusion of Liability GE Healthcare Bio Sciences Corp and its affiliates shall have no liability to an End User including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners NanoDrop is a registered trademark of NanoDrop Technologies LLC Agilent and Bioanalyzer are trademarks of Agilent Technologies Inc Affymetrix and GeneChip are registered trademarks of
17. alts and cDNA unincorporated dNTPs This step prepares the cDNA for fragmentation and labeling Before beginning the cDNA purification e Preheat the bottle of Elution Solution to 50 to 58 C for at least 10 min Make sure to add ethanol to the bottle of Nucleic Acid Wash Solution Concentrate before use This solution is referred to as Nucleic Acid Wash Solution Make sure that the Nucleic Acid Binding Buffer Concentrate is completely dissolved If not warm the solution to lt 50 C until the concentrate is solubilized Vortex the Nucleic Acid Binding Beads vigorously before use to ensure they are fully dispersed 1 Prepare the cDNA Binding Mix for the experiment At room temperature prepare the cDNA Binding Mix in a nuclease free tube for all the samples in the experiment following the instructions in the table below Note Prepare only the amount needed for all samples in the experiment plus 10 excess volume to compensate for pipetting losses Ambion WT Expression Kit User Guide 25 Ambion WT Expression Kit Procedure cDNA Binding Mix component Volume for one reaction pL Nucleic Acid Binding Beads 10 Nucleic Acid Binding Buffer Concentrate 50 1 Add 18 uL of nuclease free water and 60 uL of cDNA Binding Mix to each sample a Add 18 uL of nuclease free water to each sample for a final volume of 60 uL b Add 60 uL of cDNA Binding Mix to each sample Pipette up down 3 times to mix c Trans
18. ample results in low levels of appropriately sized cRNA CDNA product The total RNA integrity is partially degraded thereby generating short cDNA fragments Assess the integrity of your total RNA sample by determining the size of the 18S and 28S rRNA bands and the relative abundance of 28S to 18S rRNA Refer to Evaluate RNA integrity on page 17 The mRNA content of your total RNA sample is lower than expected Verify the mRNA content of your total RNA Note In healthy cells mRNA constitutes 1 to 10 of total cellular RNA Johnson 1974 Sambrook and Russel 2001 Ambion WT Expression Kit User Guide 29 Fi Ambion9 WT Expression Kit Troubleshooting 30 Ambion WT Expression Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the
19. c to collect the mix at the bottom of the tube then proceed immediately to the next step c Transfer 30 uL of the IVT Master Mix to each 60 uL Second Strand cDNA sample Mix thoroughly by gently vortexing then centrifuge briefly to collect the reaction at the bottom of the tube plate 2 Incubate for 16 hr at 40 C then overnight at 4 C a Incubate the IVT reaction for 16 hr at 40 C then overnight at 4 C in a thermal cycler using the In Vitro Transcription cRNA Synthesis method for thermal cycling shown in Table 1 on page 14 b After the incubation centrifuge briefly 5 sec to collect the reaction at the bottom of the tube plate 3 Place the cRNA on ice briefly or freeze immediately Place the reaction on ice then proceed to the next section Purify CRNA or immediately freeze the samples at 20 C for overnight storage STOPPING POINT Samples can be stored overnight at 20 C Purify cRNA In this procedure enzymes salts inorganic phosphates and unincorporated nucleotides are removed to improve the stability of the cRNA Before beginning the cRNA purification e Preheat the bottle of Elution Solution to 50 to 58 C for at least 10 min Add 100 ethanol ACS reagent grade or equivalent to the bottle of Nucleic Acid Wash Solution Concentrate before use This solution is referred to as Nucleic Acid Wash Solution 20 Ambion WT Expression Kit User Guide Ambion WT Expression Kit Procedure Ma
20. d Elution Solution y Place the cRNA on ice briefly or freeze immediately 5 Assess cRNA yield and size distribution Use 1 5 uL of cRNA to assess yield by UV absorbance Use 1 uL of cRNA to assess size distribution A240 290 A290 Ratio Frequency nm Synthesize 2nd cycle cDNA Combine 10 ug of cRNA and the Random Primers Antisense CRNA M gsm Random Primers dN6 ncu bate dUTP dNTP mix 5 a 2 Prepare the Znd Cycle Master Mix on ice and add to is NNN 5 dUTP NN each sample Incubate Ambion WT Expression Kit User Guide 11 Ambion WT Expression Kit Day 2 workflow continued Day 2 workflow continued Hydrolyze using RNase H Add RNase H to the 2nd Cycle cDNA Antisense cRNA as Incubate RNase H JL cDNA j m Optional stopping point Purify 2nd cycle cDNA Prepare the cDNA Binding Mix add to each sample GENA and transfer to a U bottom plate 3 Add ethanol to each sample and shake gently 7 Capture the Nucleic Acid Binding Beads and discard the supernatant Wash twice with Nucleic Acid Wash Solution Elute the cDNA with preheated Elution Solution Place the cDNA on ice briefly or freeze immediately 5 Nucleic Acid Binding Beads Enzyme salt phosphates unincorporated dNTP 5 TT l Assess cDNA yield and size distribution Determine cDNA yield by UV absorbance Optional Use 1 uL of cDNA to assess size
21. e Required materials Equipment Reagents and supplies Ambion WT Expression Kit Required materials Item Source Agilent 2100 Bioanalyzer Agilent Technologies Inc Ambion Magnetic Stand 96 Life Technologies Part no AM10027 Applied Biosystems Veriti 96 Well Thermal Cycler Life Technologies Part no 4375786 Barnstead Titer Plate Shaker Barnstead Lab Line Major Laboratory Supplier MLS Thermo Scientific NanoDrop ND 8000 UV Vis Spectrophotometer or Thermo Scientific NanoDrop ND 1000 UV Vis Single Channel Spectrophotometer NanoDrop Technologies Microcentrifuge with adapter for PCR 8 strip tubes or for 96 well plates MLS Item Source GeneChip WT Terminal Labeling and Controls Kit For a list of kit components go to www affymetrix com and search for Ambion WT Expression Kit Affymetrix Part no 901524 30 reaction Affymetrix Part no 901525 10 reaction Optional Quant iT RiboGreen RNA Reagent Life Technologies Optional Quant iT PicoGreen RNA Reagent Life Technologies 100 Ethanol ACS grade or equivalent MLSt 100 Isopropanol ACS grade or equivalent MLS RNA 6000 Nano Kit Agilent Technologies Inc Part no 5067 1511 Optional RNaseZap RNase Decontamination Solution Life Technologies Part no AM9780 250 mL Life Technologies Part no AM9784 4 0 L Optional Th
22. e RNA Storage Solution Life Technologies Part no AM7000 10x1 0 mL Life Technologies Part no AM7001 50 mL t Forthe MSDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all relevant precautions Ambion WT Expression Kit User Guide Ambion WT Expression Kit Day 1 workflow Day 1 workflow Synthesize first strand cDNA Prepare the First Strand Master Mix Total RNA 5 ED lt 4 oro Add the total RNA NNN NNN A eui 5 Incubate 3 Synthesize second strand cDNA Prepare the Second Strand Master Mix and cDNA add to sample 3 A AREE 4 DNA Polymerase e RNase H Incubate DNE 4 Synthesize cRNA using in vitro transcription u Prepare the IVT Master Mix and add to second dscDNA strand cDNA sample 5 NN EDEN Incubate for 16 hours TERNA Royers Antisense CRNA Stopping point wo 5 10 Ambion WT Expression Kit User Guide Ambion WT Expression Kit Day 2 workflow Day 2 workflow Purify cRNA Prepare the cRNA Binding Mix and add to each Antisense cRNA sample E Nucleic Acid Binding Beads Add 100 isopropanol to each sample and shake NN gently unincorporated dNTP 3 y Capture the Nucleic Acid Binding Beads and discard the supernatant y Wash twice with Nucleic Acid Wash Solution y Elute cRNA with preheate
23. e liquid at the bottom of the tube Ambion9 WT Expression Kit User Guide Evaluate RNA quality Evaluate RNA integrity Synthesize first strand cDNA Ambion WT Expression Kit r Procedure 3 Add 2 uL of the first dilution to 98 uL of Poly A Control Dil Buffer to prepare the second dilution 1 50 4 Mix thoroughly and spin down to collect the liquid at the bottom of the tube 5 Add 2 uL of the second dilution to 98 uL of Poly A Control Dil Buffer to prepare the third dilution 1 50 6 Mix thoroughly and spin down to collect the liquid at the bottom of the tube 7 Add 2 uL of the third dilution to 18 uL of Poly A Control Dil Buffer to prepare the fourth dilution 1 10 8 Mix thoroughly and spin down to collect the liquid at the bottom of the tube 9 Add 2 uL of this fourth dilution to 100 ng of total RNA Note The first dilution of the Poly A RNA controls can be stored up to 6 weeks in a non frost free freezer at 20 C and frozen thawed up to eight times RNA quality affects how efficiently an RNA sample is amplified using this kit High quality RNA is free of contaminating proteins DNA phenol ethanol and salts To evaluate RNA quality determine its A569 A go ratio RNA of acceptable quality is in the range of 1 7 to 2 1 The integrity of the RNA sample or the proportion that is full length is an important component of RNA quality Reverse transcribing partially degraded mRNA may generate cDNA that lac
24. ession Kit User Guide 13 Ambion WT Expression Kit Procedure Note Concentration fluctuations that are caused by condensation can affect your yield Ensure that the heated lid feature of the thermal cycler is working properly Table 1 Thermal cycling methods Heated lid Alternate Method temp Protocol Step 1 Step 2 Step 3 Step 4 First Strand cDNA BMC 105 C 25 C 60 min 42 C 60 min 49C 2 min Synthesis Second Strand cDNA RT or Lid open 16 C 60 min 65 C 10 min 4 C 2 min u Synthesis disable In Vitro Transcription 509C 40 C oven 40 C 16 hrs 4 C Hold E CRNA Synthesis 2nd Cycle cRNA 75 C 105 C 70 C 5 min 25 C 5 min 4 C 2 min _ Denaturation 2nd Cycle cDNA PSIG 105 C 25 C 10 min 42 C 90 min 70 C 10 min 4 C 2min Synthesis RNase H Hydrolysis 75 C 105 C 37 C 45 min 95 C 5 min 4 C 2 min t For thermal cyclers that lack a programmable heated lid Prepare the Control RNA and total RNA 14 Prepare the Control RNA Note For this section review the safety information on page 31 To verify that the reagents are working as expected a Control RNA sample 1 mg mL total RNA from HeLa cells is included with the kit To prepare the control 1 Dispense 2 uL of Control RNA in 38 uL of nuclease free water for a total volume of 40 uL 2 Follow the Synthesize first strand cDNA on page 17 but in Step 2 use 1 uL of diluted Control RNA 50 ng in the control re
25. fer each sample to a well of a U bottom plate 2 Add 120 uL of ethanol to each sample then shake gently for 2 min a Add 120 uL of 100 ethanol to each sample Pipette up down 3 times to mix IMPORTANT Add ethanol in this step Do not use isopropanol b Gently shake for 22 min to thoroughly mix setting 4 on the Lab Line Titer Plate Shaker The cDNA in the sample binds to the Nucleic Acid Binding Beads during this incubation 3 Capture the Nucleic Acid Binding Beads then discard the supernatant a Move the plate to a magnetic stand to capture the magnetic beads When the capture is complete after 5 min the mixture is transparent and the Nucleic Acid Binding Beads form pellets against the magnets in the magnetic stand The exact capture time depends on the magnetic stand that you use b Carefully aspirate and discard the supernatant without disturbing the magnetic beads then remove the plate from the magnetic stand 4 Wash twice with 100 uL of Nucleic Acid Wash Solution IMPORTANT Make sure that ethanol is added to the bottle of Nucleic Acid Wash Solution Concentrate before using it a Add 100 uL of Nucleic Acid Wash Solution to each sample then shake the samples at moderate speed for 1 min setting 7 on the Lab Line Titer Plate Shaker Note Although the Nucleic Acid Binding Beads may not fully disperse during this step this does not affect DNA purity or yield b Move the plate to a magnetic stand to capture t
26. h purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing dlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product This product or portions thereof is manufactured and sold under license from GE Healthcare End User Terms And Conditions Acceptance These terms and conditions shall govern the purchase use transfer and acceptance of the products described in the purchase order quotation or invoice which products are sold and distributed by Applera Corporation to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly conditional upon End User s acceptance of these terms and conditions Restrictions on Use End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand along product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE He
27. he Nucleic Acid Binding Beads as in step 3 c Carefully aspirate and discard the supernatant without disturbing the Nucleic Acid Binding Beads then remove the plate from the magnetic stand d Repeat step 5a to step 5c to wash a second time with 100 uL of Nucleic Acid Wash Solution e Move the plate to a shaker then shake the plate vigorously for 1 min to evaporate residual ethanol from the beads setting 10 on the Lab Line Titer Plate Shaker Dry the solution until no liquid is visible but the pellet appears shiny Additional time may be required Do not over dry the beads 26 Ambion WT Expression Kit User Guide Assess cDNA yield and size distribution 5 Ambion WT Expression Kit Procedure Elute cDNA with 30 uL of preheated Elution Solution a Elute the purified cDNA from the Nucleic Acid Binding Beads by adding 30 uL of preheated 55 to 58 C Elution Solution to each sample Incubate for 2 min at room temp without shaking b Vigorously shake the plate for 3 min setting 10 on the Lab Line Titer Plate Shaker Make sure that the Nucleic Acid Binding Beads are fully dispersed If they are not continue shaking until the beads are dispersed and or pipette up and down 3 times c Move the plate to a magnetic stand to capture the Nucleic Acid Binding Beads d Transfer the supernatant which contains the eluted cDNA to a nuclease free multiwell plate 6 Place the cDNA on ice briefly or freeze immediately
28. ke sure that the Nucleic Acid Binding Buffer Concentrate is completely dissolved If not warm the solution to lt 50 C until the concentrate is solubilized e Vortex the Nucleic Acid Binding Beads vigorously before use to ensure that they are fully dispersed 1 Prepare the cRNA Binding Mix Note For this section review the safety information on page 31 At room temperature immediately before use prepare the cRNA Binding Mix in a nuclease free tube for all the samples in the experiment Follow the instructions in the table below IMPORTANT Prepare only the amount that is needed for all samples in the experiment plus 10 excess volume to compensate for pipetting losses cRNA Binding Mix component Volume for one reaction pL Nucleic Acid Binding Beads 10 Nucleic Acid Binding Buffer Concentrate 50 2 Add 60 uL of cRNA Binding Mix to each sample a Add 60 uL of cRNA Binding Mix to each sample Pipette up down 3 times to mix b Transfer each sample to a well of a U Bottom Plate 3 Add 60 uL of isopropanol to each sample then shake gently for 2 min a Add 60 uL of 100 isopropanol to each sample Pipette up down 3 times to mix IMPORTANT Add isopropanol at this step Do not use ethanol b Gently shake for 22 min to thoroughly mix setting 4 on the Lab Line Titer Plate Shaker The cRNA in the sample binds to the Nucleic Acid Binding Beads during this incubation 4 Capture the Nucleic Acid
29. ks parts of the coding region Two methods to evaluate RNA integrity are Microfluidic analysis using the Agilent 2100 Bioanalyzer with an RNA LabChip Kit Denaturing agarose gel electrophoresis With microfluidic analysis you use the RNA Integrity Number RIN to evaluate RNA integrity For more information on how to calculate RIN go to www chem agilent com With denaturing agarose gel electrophoresis and nucleic acid staining you separate and make visible the 285 and 185 rRNA bands The mRNA is likely to be full length if the e 285 and 185 rRNA bands are resolved into two discrete bands that have no significant smearing below each band e 285 rRNA band intensity is approximately twice that of the 185 rRNA band In this reverse transcription procedure total RNA is primed with engineered primers containing a T7 promoter sequence The reaction synthesizes single stranded cDNA containing a T7 promoter sequence Note To include premixed controls from the Affymetrix GeneChip Poly A RNA Control Kit Part no 900433 or equivalent add the reagents to the total RNA samples See Prepare the Poly A RNA Controls on page 15 for more information 1 Prepare a First Strand Master Mix then dispense 5 uL into a reaction tube plate Ambion WT Expression Kit User Guide 17 Procedure Ambion WT Expression Kit Note For this section review the safety information on page 31 IMPORTANT Prepare master mixes for each step of
30. mple yield low levels of amplified cRNA product or low levels of appropriately sized cRNA product Incubation temperatures are incorrect or inaccurate Calibrate your thermal cycler Condensation formed in the tubes during the incubations Check that the heated lid is working correctly and is set to the appropriate temperature cRNA purification not performed properly Perform the purification as described in this document and make sure that you use the correct alcohol Pipettes tubes and or equipment are contaminated with nuclease Remove RNases and DNases from surfaces using Ambion RNaseZap RNase Decontamination Solution The positive control sample produces expected results but your total RNA sample results in low levels of amplified cRNA cDNA product The input total RNA concentration is lower than expected Repeat the A gg reading of your RNA sample Use 100 to 200 ng of total RNA in the first strand cDNA synthesis procedure Your input RNA contains contaminating DNA protein phenol ethanol or salts causing inefficient reverse transcription Phenol extract and ethanol precipitate your total RNA or use the Ambion MEGAclear Kit Part no AM1908 28 Ambion WT Expression Kit User Guide Ambion WT Expression Kit Troubleshooting Observation Possible cause Solution The positive control sample produces expected results but your total RNA s
31. ncial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 en Ambion WT Expression Kit User Guide 33 Appendix Safety Bi
32. nscription The reaction uses DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA 1 Prepare a Second Strand Master Mix then add 50 uL to each sample Note For this section review the safety information on page 31 a On ice prepare the Second Strand Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare the master mix for all the first strand synthesis cDNA samples in the experiment Include 5 excess volume to correct for pipetting losses Second Strand Master Mix Volume for one reaction component uL Nuclease free Water 32 5 uL Second Strand Buffer Mix 12 5 uL Second Strand Enzyme Mix 5 uL Total Volume 50 uL b Mix thoroughly by gently vortexing Centrifuge briefly 5 sec to collect the mix at the bottom of the tube and proceed immediately to the next step c Transfer 50 uL of the Second Strand Master Mix to each 10 uL first strand synthesis cDNA sample Mix thoroughly by gently vortexing or flicking the tube 3 or 4 times Centrifuge briefly to collect the reaction at the bottom of the tube plate and proceed immediately to the next step 2 Incubate for 1 hr at 16 C then for 10 min at 65 C then for at least 2 min at 4 C a Incubate for 1 hr at 16 C then for 10 min at 65 C then for at least 2 min at 4 C in a thermal cycler using the Second Strand cDNA Synthesis method for thermal cycling that i
33. o a shaker and shake the plate vigorously for 1 min to evaporate residual ethanol from the beads setting 10 on the Lab Line Titer Plate Shaker Dry the solution until no liquid is visible yet the pellet appears shiny Additional time may be required Do not over dry the beads 6 Elute cRNA with 40 uL of preheated Elution Solution a Add to each sample 40 uL of preheated 55 to 58 C Elution Solution to elute the purified cRNA from the Nucleic Acid Binding Beads Incubate without shaking for 2 min b Vigorously shake the plate for 3 min setting 10 on the Lab Line Titer Plate Shaker then check to make sure that the Nucleic Acid Binding Beads are fully dispersed If they are not continue shaking until the beads are dispersed and or pipette up down 3 times Pellets can be disrupted by manual pipetting using a single channel P 200 or equivalent c Move the plate to a magnetic stand to capture the Nucleic Acid Binding Beads d Transfer the supernatant which contains the eluted cRNA to a nuclease free multiwell plate 7 Place the cRNA on ice briefly or freeze immediately Place the reaction on ice then proceed immediately to the Synthesize 2nd cycle cDNA on page 23 or freeze the samples at 20 C for overnight storage STOPPING POINT Samples can be stored overnight at 20 C Assess cRNA yield Expected cRNA yield and Slee The cRNA yield depends on the amount and quality of poly A RNA in the input total distribu
34. of sizes from 50 to 4500 nt with most of the cRNA sizes in the 200 to 2000 nt range The distribution is quite jagged and does not resemble the profile observed when using a traditional dT based amplification kit such as a MessageAmp kit This step is optional 1 Optional Determine cRNA size distribution using a Bioanalyzer Instrument We recommend analyzing cRNA size distribution using an Agilent 2100 Bioanalyzer Instrument a RNA 6000 Nano Kit Part no 5067 1511 and mRNA Nano Series II assay If there is sufficient yield then load approximately 300 ng of cRNA per well on the Bioanalyzer Instrument If there is not sufficient yield then use as little as 200 ng of cRNA per well To analyze cRNA size using a Bioanalyzer Instrument follow the manufacturer s instructions Synthesize 2nd In this procedure sense strand cDNA is synthesized by the reverse transcription of cycle cDNA cRNA using random primers The sense strand cDNA contains dUTP at a fixed ratio relative to dTTP 10 ug of cRNA is required for 2nd cycle cDNA synthesis 1 Prepare 10 ug of CRNA in a volume of 22 uL On ice prepare 455 ng uL cRNA This is equal to 10 ug cRNA in a volume of 22 uL If necessary use nuclease free water to bring the cRNA sample to 22 uL If the cRNA is too dilute then concentrate the cRNA by vacuum centrifugation If vacuum centrifugation is required for one sample then we recommend drying all of the samples to reduce sam
35. ological hazard safety 34 Ambion WT Expression Kit User Guide Bibliography Johnson L F Abelson H T Green H and S Penman 1974 Cell 1 95 100 Sambrook J and D W Russel 2001 Extraction purification and analysis of mRNA from eukaryotic cells In Molecular cloning a laboratory manual third edition Vol 1 Cold Spring Harbor New York Cold Spring Harbor Press Van Gelder R N von Xastrow M E Yool E et al 1990 Proc Natl Acad Sci USA 87 1663 1667 Ambion WT Expression Kit User Guide 35 60Z7SCbb Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support technologies
36. ple to sample variability To concentrate the samples place 10 ug of the sample in a tube bring to a set volume of 30 uL using Nuclease free water dry the samples to 15 uL then bring the samples to a final volume of 22 uL using Nuclease free water IMPORTANT During vacuum centrifugation check the progress of drying every 5 to 10 min Remove the sample from the concentrator when it reaches the desired volume Avoid drying cRNA samples to completion Ambion WT Expression Kit User Guide 23 Ambion WT Expression Kit Procedure 2 Combine 10 ug of cRNA and the Random Primers IMPORTANT Prepare master mixes for each step of the procedure to save time improve reproducibility and minimize pipetting error Before preparing the master mixes inspect the buffer mixes for precipitates If necessary warm the buffer mix es at 55 C for 1 to 2 min or until the precipitate is fully dissolved When preparing the master mixes thoroughly vortex each buffer mix before use Mix the enzymes by gently flicking the tubes a few times before use Keep the buffer mixes at room temperature until needed to prevent re precipitation Keep the enzyme mixes on ice at all times a On ice using supplied PCR tubes or plate combine e 22 uL of cRNA 10 ug e 2 uL of Random Primers b Mix thoroughly by gently vortexing Centrifuge briefly 5 sec to collect the reaction at the bottom of the tube plate Place on ice 3 Incubate for 5 min at 70 C
37. pliy 4 0 souder albaranes 35 Ambion WT Expression Kit User Guide About This Guide CAUTION ABBREVIATED SAFETY ALERTS Hazard symbols and hazard types specified in procedures may be abbreviated in this document For the complete safety information see the Safety appendix in this document IMPORTANT Before using this product read and understand the information the Safety appendix in this document Purpose The Ambion WT Expression Kit User Guide provides detailed information for preparing RNA samples for Affymetrix whole transcriptome microarray analysis using the Ambion WT Expression Kit User attention words Five user attention words appear in this document Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation or accurate chemistry kit use CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury Except for IMPORTANTS the safety alert word
38. rols to your RNA the volume of RNA must be less than 3 uL If necessary use a Speed Vac or ethanol precipitation to concentrate the RNA samples For example when performing the control RNA reaction combine 1 uL of RNA 50 ng uL 2 uL of diluted Poly A Spike Controls and 2 uL of nuclease free water b Mix thoroughly by gently vortexing Centrifuge briefly to collect the reaction at the bottom of the tube plate then proceed immediately to the next step 3 Incubate for 1 hour at 25 C then for 1 hour at 42 C then for at least 2 min at 4 C a Incubate for 1 hr at 25 C then for 1 hr at 42 C then for at least 2 min at 4 C in a thermal cycler using the First Strand cDNA Synthesis Method that is shown in Table 1 on page 14 18 Ambion WT Expression Kit User Guide Ambion WT Expression Kit Procedure b Immediately after the incubation centrifuge briefly 5 sec to collect the first strand cDNA at the bottom of the tube plate Place the sample on ice for 2 min to cool the plastic then proceed immediately to the next section Synthesize second strand cDNA IMPORTANT Transferring Second Strand Master Mix to hot plastics may significantly reduce cRNA yields Holding the First Strand cDNA Synthesis reaction at 4 C for longer than 10 minutes may significantly reduce cRNA yields Synthesize second In this procedure single stranded cDNA is converted to double stranded cDNA strand cDNA which acts as a template for tra
39. s in user documentation appear with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to the instrument See the Safety appendix for descriptions of the symbols Ambion WT Expression Kit User Guide 5 About This Guide User attention words 6 Ambion WT Expression Kit User Guide Ambion WT Expression Kit Product information Purpose of the The Ambion WT Expression Kit enables you to prepare RNA samples for Affymetrix product whole transcriptome microarray analysis The kit generates sense strand cDNA from total RNA for fragmentation and labeling using the Affymetrix GeneChip WT Terminal Labeling Kit Part no 900671 The kit is optimized for use with human mouse and rat Affymetrix GeneChip Sense Target ST Arrays The WT Expression Kit uses a reverse transcription priming method that specifically primes non ribosomal RNA from your sample including both poly A and non poly A mRNA Primers that avoid rRNA binding are designed by using a proprietary oligonucleotide matching algorithm These primer sequences provide complete and unbiased coverage of the transcriptome while significantly reducing the priming of rRNA Ambion WT Expression Kit User Guide 7 Ambion WT Expression Kit Product information Kit contents and storage For manual use only For high throughput automated use only
40. s shown in Table 1 on page 14 IMPORTANT Disable the heated lid of the thermal cycler or keep the lid off during the second strand cDNA synthesis b Immediately after the incubation centrifuge briefly 5 sec to collect the double stranded cDNA at the bottom of the tube plate Place the sample on ice to cool the plastic then immediately proceed to the next section Synthesize CRNA by In Vitro Transcription Ambion WT Expression Kit User Guide 19 Ambion WT Expression Kit Procedure Synthesize cRNA In this procedure antisense cRNA is synthesized and amplified by in vitro by In Vitro transcription IVT of the second strand cDNA template using 17 RNA polymerase Transcription This method of RNA sample preparation is based on the original T7 in vitro transcription technology known as the Eberwine or RT IVT method Van Gelder et al 1990 1 Prepare an IVT Master Mix then dispense 30 uL to each sample Note For this section review the safety information on page 31 a At room temperature prepare an IVT Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Prepare master mix for all the second strand cDNA 60 uL samples Include 5 excess volume to correct for pipetting losses IVT Master Mix Volume for one reaction component pL IVT Buffer Mix 24 IVT Enzyme Mix 6 Total Volume 30 b Mix thoroughly by gently vortexing Centrifuge briefly 5 se
41. the procedure to save time improve reproducibility and minimize pipetting error Before preparing the master mixes inspect the buffer mixes for precipitates If necessary warm the buffer mix es at 55 C for 1 to 2 min or until the precipitate is fully dissolved When preparing the master mixes thoroughly vortex each buffer mix before use Mix the enzymes by gently flicking the tubes a few times before use Keep the buffer mixes at room temperature until needed to prevent re precipitation Keep the enzyme mixes on ice at all times a Atroom temperature prepare the First Strand Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Include 5 excess volume to correct for pipetting losses First Strand Master Mix component yolume na reaction First Strand Buffer Mix 4 First Strand Enzyme Mix 1 Total Volume 5 b Mix thoroughly by gently vortexing Centrifuge briefly 5 sec to collect the mix at the bottom of the tube Proceed immediately to the next step c Transfer 5 uL of the First Strand Master Mix to the supplied PCR tubes 2 Add 5 uL of total RNA mix thoroughly by gently vortexing centrifuge briefly then proceed immediately to the next step a Add 5 uL RNA to each tube or well containing the First Strand Master Mix for a final reaction volume of 10 uL If necessary use nuclease free water to bring the RNA to 5 uL Note If you are adding Poly A Spike Cont
42. then 5 min at 25 C then 2 min at 4 C a Incubate in a thermal cycler using the 2nd Cycle cRNA Denaturation method for thermal cycling that is shown in Table 1 on page 14 b After the incubation centrifuge briefly 5 sec to collect the 2nd Cycle cDNA at the bottom of the tube plate 4 Prepare the 2nd Cycle Master Mix on ice then add 16 uL to each sample a At room temperature prepare the 2nd Cycle Master Mix in a nuclease free tube Combine the components in the sequence shown in the table below Include 5 excess volume to correct for pipetting losses 2nd Cycle Master Mix Volume for one reaction component uL 2nd Cycle Buffer Mix 8 2nd Cycle Enzyme Mix 8 Total Volume 16 b Mix thoroughly by gently vortexing Centrifuge briefly 5 sec to collect the mix at the bottom of the tube plate Proceed immediately to the next step c Transfer 16 uL of 2nd Cycle Master Mix to each 24 uL cRNA Random Primer sample Mix thoroughly by gently vortexing Centrifuge briefly to collect the reaction at the bottom of the plate tube Proceed immediately to the next step 5 Incubate for 10 min at 25 C then 90 min at 42 C then 10 min at 70 C then for at least 2 min at 4 C a Incubate in a thermal cycler using the 2nd Cycle cDNA Synthesis method for thermal cycling that is shown in Table 1 on page 14 IMPORTANT Cover the reactions with the heated lid at 75 C 24 Ambion WT Expression Kit User Guide Ambion
43. tion RNA Because the proportion of poly A RNA in total RNA is affected by factors such as the health of the organism and the organ from which it is isolated CRNA yield from equal amounts of total RNA may vary considerably During development of this kit using a wide variety of tissue types 50 ng of input total RNA yielded 15 to 50 ug of cRNA For most tissue types the recommended 100 ng of input total RNA should provide gt 20 ug of cRNA 1 Determine cRNA yield by UV absorbance Determine the concentration of a cRNA solution by measuring its absorbance at 260 nm We recommend evaluating the absorbance of 1 5 uL of cRNA sample using a NanoDrop Spectrophotometer 22 Ambion WT Expression Kit User Guide Ambion WT Expression Kit r Procedure Alternatively determine the cRNA concentration 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 Calculate the concentration in pg mL using the equation shown below 1 Ax 69 40 ug RNA mL A goxdilution factorx40 ug RNA mL Optional Determine cRNA yield using Quant iT RiboGreen RNA Reagent If a fluorometer or a fluorescence microplate reader is available use the Quant iT RiboGreen RNA Reagent Invitrogen to measure RNA concentration Follow the manufacturer s instructions Optional Expected cRNA size distribution The expected cRNA profile is a distribution
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