User Manual-ENZ-42421-100 - Rev 1.1.0 April 2010.pub
Contents
1. Reaction tube conden sation Briefly centrifuge the reaction tube s Mix gently and move the reaction tube s to an incubator where condensation does not occur RNA sample vacuum centrifuged to dryness Monitor sample volume during vacuum centrifugation to avoid drying to com pletion RNA is difficult to resuspend if dried completely Inadequate elution of cDNA Heat elution buffer to 70 C Proper maintenance of temperature at this step is critical for maximal elution of cDNA from the column filter Inadequate elution of aRNA Elute nucleic acids from column filter purification systems with enough elu tion solution to completely saturate the filter Dispense the elution buffer in the center of the column bed Incorrect spectropho tometer readings Check the accuracy of your spectro photometer Alternatively use another method for quantitation of aRNA such as staining with a sensitive RNA dye Average size of con trol aRNA is greater than 1 kb but aver age size of experi mental aRNA is less than 0 5 kb RNA is partially de graded Prepare fresh samples following the guidelines in Appendix A to prevent RNase contamination Use nuclease free plasticware and decontaminate pipettes and work surfaces with com mercially available RNase decontami nation solutions 12 VIII Troubleshooting Guide Control RNA Reaction High quality Control RNA 100 ng uL Cat No2. 42 Pause Reverse Transcription ESI Len mintes Second Strand Synthesis pa Uu as In Vitro Transcription ya 16 noue Gaast Ema C cDNA AND aRNA PURIFICATION GUIDELINES There are several commercially available column filter based purifi cation systems for the purification of the aRNA To avoid precipita tion of reagents or sample ensure that all solutions are at room tem perature and thoroughly mixed before use All excess wash solution must be removed from the column filter prior to aRNA elution The 100 Reaction Single Round RNA Amplification and Labeling System is compatible with the Agencourt BioScience RNAClean purification system and is recommended using the following proto cols l cDNA 1 Equilibrate the RNAClean reagents to room temperature and then resuspend the magnetic beads during the last 30 min of second strand cDNA synthesis 2 Add 135 uL of RNAClean to each well of a 96 well U bottom plate Add 75 uL of each second strand reaction to the plate containing beads and incubate for 5 minutes at room temperature 3 Separate by placing the 96 well plate on the magnetic stand for 10 minutes or until the mixture is clear 4 Gently remove the supernatant while keeping the plate on the magnetic stand 5 Repeat Steps 1 4 with the remaining second strand reaction volume 6 Remove the plate from the magnetic stand and wash the beads with 200 uL of 70 ethanol freshly prepared and incubate for 30
3. at 42 C for 2 hours Second Strand cDNA Synthesis ee el AAAA T7 3 Add 130 uL Second Strand Master Mix adiac mra TITT T7 Incubate at 16 C for 2 hours 4 cDNA Purification Note This kit does not include consumables or reagents ef CO NL eren UUUUU 5 required for purification Please refer to the Appendix for ry suggestions and guidelines 7 UUUUU 5 ee Ts In Vitro Transcription and Biotin Labeling n e e Add 27 uL IVT Master Mix i ee yuuuy s Incubate at 37 C for 6 hours for input RNA gt 2 ug ee Incubate at 37 C for 16 hours for input RNA 0 25 2 ug 4 aRNA Purification Fragmentation and Array Hybridization D ANALYSIS OF BIOTIN LABELED TRANSCRIPT The RNA yield can be estimated by absorbance detection at 260 nm and 280 nm using a UV Vis spectrophotometer If using a Nano Drop Spectrophotometer assay 2 uL of the aRNA sample directly If analyzing by Agilent 2100 Bioanalyzer or similar method the distri bution of biotin labeled aRNA should range from 200 6000 nt with a broad peak between 1 kb and 2 kb Figure 1 If a bioanalyzer is not available the quality of the amplified aRNA can be assessed by gel electrophoresis For high quality total RNA the resulting aRNA appear as a smear of RNAs ranging from 200 nt to 5000 nt with and average size of approximately 1200 nt Figure 1 Analysis of biotin labeled aRNA using the Single Round RNA Amplification and Labeling System After puri
4. seconds at room temperature 7 Separate by placing the 96 well plate on the magnetic stand for approximately 5 minutes or until the mixture is clear 8 Aspirate and discard the supernatant and repeat steps 6 7 Aspirate trace amounts of ethanol without disturbing the beads 9 Remove the plate from the magnetic stand and allow the beads to air dry for 10 minutes 10 Add 36 uL of RNase free water 25 C to each well and gen tly shake on orbital shaker for 2 minutes 11 Separate the beads by applying the magnet for 10 minutes or until clear 12 Collect and transfer 33 uL of cDNA solution to a 96 well PCR plate 13 Purified cDNA can be stored at 20 C for up to 3 days before use VI Appendices A RECOMMENDATIONS FOR RNASE FREE TECHNIQUE Avoid all sources of RNase contamination as generation of high quality labeled aRNA product requires that full length RNA be main tained from sample preparation to cDNA synthesis and transcription Wear disposable powder free gloves and change frequently throughout Avoid RNase containing surfaces Use only the reagents supplied in this kit Other reagents may not be compatible Use only sterile DNase and RNase free pipet tips microcentrifuge tubes and other plasticware Clean and decontaminate pipettes and work surfaces with commer cially available RNase decontaminating solutions or wipes RNA PREPARATION GUIDELINES RNA purity and integrity affect both the quant
5. 72 440 655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com For research use only Rev 1 1 0 April 2010 Notice to Purchaser This product is manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit this product commercially Any commercial use development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty This product is offered under a limited warranty The product is guaranteed to meet appropriate specifications described in the package insert at the time of shipment Enzo Life Sciences sole obligation is to replace the product to the extent of the purchase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo is a trademark of Enzo Life Sciences Inc Several of Enzo s products and product applications are covered by US and foreign patents and patents pending Problem Potential Cause Suggestion Low yields with both experimental sam ples and control RNA continuation Incorrect incubation temperatures Check the accuracy of the tempera tures of your incubators and thermal cycler
6. ENZ 42421 CR can be used to help evaluate kit performance for troubleshooting and as an aid to first time users Parallel reactions with sample and control RNA 5 uL 500 ng can be performed Typical yield of purified aRNA generated from a 16 hr in vitro transcription reaction with 500 ng of input control RNA in the first strand cDNA synthesis reaction should be 2 30 ug In addition 100 ng of the aRNA generated from control RNA can be ana lyzed on a bioanalyzer and results compared to the profile in Figure 1 Appendix C Problem Potential Cause Suggestion Low aRNA yields with experimental samples Organic protein or salt contaminants in the input RNA Perform parallel reaction with control RNA ENZ 42406 CR sold separately Use 5 uL 500 ng in the first strand synthesis reaction Typical yield from this input is 30 ug or more If control RNA amplifies to this level but not your experimental samples repurify your starting material Use extra caution not to carry over organic organic solvents or dena turants during purification Low mRNA content of ex perimental sample Increase the total RNA input if the experi mental RNA sample is expected to have low mRNA content Input RNA does not contain poly A tracts RNA amplification with this kit requires the use of oligo dT primers Do not substitute random or gene specific primers with this kit Degraded input RNA Prepare fresh sample following t
7. Enzo Enabling Discovery in Life Science www enzolifesciences com Enabling Discovery in Life Science 100 Reaction Single Round RNA Amplification and Biotin Labeling System Instruction Manual Cat No ENZ 42421 100 NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA T 1 800 942 0430 610 941 0430 F 610 941 9252 E info usaGenzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 441 061 926 89 89 F 441 061 926 89 79 E info chGenzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 449 0 7621 5500 527 E info deGenzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 432 03 466 04 20 F 432 03 466 0429 E info be enzolifesciences com www enzolifesciences com incorpora UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845601 1488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info ukGenzolifesciences com www enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33 4
8. RNA PREPARATION GUIDELINES eee 7 C cDNA AND aRNA PURIFICATION GUIDELINES 8 D ANALYSIS OF BIOTIN LABELED TRANSCRIPT 10 E aRNA FRAGMENTATION eceeeeetenereteen teer ette rns 10 VII References ueni eroi enn ca mee Rai arare 10 VIII Troubleshooting Guide 11 l Introduction The 100 Reaction Single Round RNA Amplification and Biotin Label ing System provides an optimized protocol and reagents for the produc tion of biotin labeled antisense RNA aRNA from total cellular RNA sam ples 0 25 ug 5 ug in less than 24 hours for array analysis The 100 Reaction Single Round RNA Amplification and Biotin Labeling System has been optimized for superior performance across several automated liquid handling platforms The result is a comprehensive system that re duces variability while subsequently improving reproducibility data quality and throughput for automated genomic environments The complete system is composed of reagents for cDNA synthesis and in vitro transcription labeling and does not provide materials required for purification ll Procedural Overview d First Strand cDNA Synthesis Add 1 uL T7 Oligo dT Primer to 0 25 5 ug total RNA Bring total volume to 13 uL with nuclease free water Incubate at 70 C for 10 minutes and move to 42 C Add 7 uL of First Strand Master Mix L MG 5 Incubate
9. ce Briefly centrifuge the vials containing enzymes immediately prior to use and place on ice 2 Prepare the in vitro transcription master mix by combining the reagents Table 6 below in a sterile nuclease free microcentri fuge tube Assemble enough master mix for all samples plus 10 overage to accommodate pipeting error Gently mix the reagents by pipetting up and down several times centrifuge and place the IVT Master Mix on ice Table 6 n Vitro Transcription IVT Master Mix Component Vial ID Vol per Reaction IVT Reaction Buffer RB 6 uL Biotin Labeled Ribonucleotide B 6 uL DTT D 6 uL Enhancer Cocktail EC 6 uL T7 RNA Polymerase T7 3 uL Total 27 uL 3 At room temperature add 27 yl of IVT Master Mix to each well containing sample 4 Mix gently by pipeting up and down several times Cover the plate with a sealing film then centrifuge briefly to collect the mix ture to the bottom of the wells 5 Immediately place the 96 well plate in a 37 C thermal cycler or heating block with heated lid and incubate for 16 hours For total RNA inputs 2 2 ug a 6 hour incubation is sufficient 6 Stop the reaction by placing the 96 well plate on ice If using a thermal cycler bring the reaction temperature to 4 C and hold 7 Proceed to aRNA purification Unpurified samples can be stored at x 70 C for up to 3 days aRNA PURIFICATION A suggested protocol for the aRNA purification is provided in Appendi
10. e beads Remove the plate from the magnetic stand and allow the beads to air dry for 10 minutes Add 62 uL of RNase free water 25 C to each well seal and vortex or shake for 1 minute Separate the beads by applying the magnet for 10 minutes or until clear Collect and transfer 60 uL of aRNA solution to a 96 well PCR plate Analyze the RNA as required or store at 20 C overnight or at 80 C for long term storage Reagents Provided and Storage Store all kit components at 20 C in a non frost free freezer Water can be left at room temperature after first thawing Table 1 Kit Components Reagents Min Vol Supplied Vial ID dNTP Mix 400 uL dN Promoter Primer 100 uL P First Strand Buffer 200 uL FB DTT 800 uL D Reverse Transcriptase 100 uL RT RNase Inhibitor 100 uL l DNA Polymerase 500 uL DP RNase H 100 uL RH Second Strand Buffer 1 5 mL SB IVT Reaction Buffer 600 uL RB 10X Biotin Labeled Ribonucleotide 600 uL B Enhancer Cocktail 600 uL EC T7 RNA Polymerase 300 uL T7 Nuclease free Water 13 mL Ww Table 2 Related Product Available Separately Reagent Supplied Cat No Control RNA 100 pg mL 10 uL ENZ 42406 CR Additional Materials Required Equipment e Thermal cycler with heated lid or heating blocks with heated lid e Cold block 4 C e Microcentrifuge e Orbital shaker for 96 well plates optional e UV Spectrophotometer optional e Bioanalyzer Agilent optional Materials e Isola
11. fication 400 ng of synthesized aRNA was evaluated using the RNA Nano Pico LabChip kit on an Agilent 2100 BioAnalyzer E aRNA FRAGMENTATION For proper hybridization to microarrays labeled aRNAs should be reduced in size Follow the guidelines of your microarray manufac turer regarding aRNA fragmentation In general any RNA hydroly sis method that generates RNA fragments in the 75 200 nucleotide size range is acceptable Vil References 1 Van Gelder et al PNAS 87 1990 1663 7 Van Gelder et al PNAS 87 1990 10 ll aRNA 1 10 11 12 13 Equilibrate the RNAClean reagents to room temperature and then resuspend the magnetic beads during the last 30 min of the IVT reaction Transfer the samples from the IVT reaction into a 96 well U bottom plate Add 108 uL of magnetic beads to each sample and incubate for 5 minutes at room temperature Separate by placing the 96 well plate on the magnetic stand for 10 minutes or until the mixture is clear Gently remove the supernatant while keeping the plate on the magnetic stand Remove the plate from the magnetic stand and wash the beads with 200 uL of 70 ethanol freshly prepared and incubate for 30 seconds at room temperature Separate by placing the 96 well plate on the magnetic stand for approximately 5 minutes or until the mixture is clear Aspirate and discard the supernatant and repeat steps 6 7 Aspirate trace amounts of ethanol without disturbing th
12. he guide lines in Appendix A to prevent RNase con tamination Use nuclease free plasticware and decontaminate pipettes and work sur faces with commercially available RNase decontamination solutions Low yields with both experimental sam ples and control RNA Inadequate thawing and mixing of reagents Ensure reagents are completely thawed with no solids remaining Gently mix and centrifuge reagents briefly Nuclease contamination Prepare fresh samples following the guidelines in Appendix A to prevent RNase contamination Use nuclease free plasticware and decontaminate pipettes and work surfaces with com mercially available RNase decontami nation solutions 11 Continued Contents l Introduction cece cessseseceseeesesnessseeseeuseenaassseuesneneaas 1 Il Procedural Overview eere 1 Ill Reagents Provided and Storage 2 IV Additional Materials Required 2 V Methods and Procedures ee 3 A FIRST STRAND SYNTHESIS eeeeeeeees 4 B SECOND STRAND SYNTHESIS e 5 C CDNA PURIFICATION ieeeseeeeeeeter eterne tree ettet 5 D RNA TRANSCRIPT LABELING eeeeeeeneteeeteec 6 E aRNA PURIFICATION eene 6 MI Appendices 5 2 ei anarei daaa de 7 A RECOMMENDATIONS FOR RNASE FREE TECHNIQUE 7 B
13. hortly before the completion of the first strand synthesis reaction prepare the Second Strand Master Mix Table 5 below in a sterile nuclease free microcentrifuge tube Assemble enough master mix for all samples plus 10 overage to accommodate pipeting error Gently mix the reagents by pipetting up and down several times centrifuge and place the Second Strand Master Mix on ice Table 5 Second Strand Master Mix Component Vial ID Vol per Reaction Nuclease free Water Ww 106 uL dNTP Mix dN 3 uL Second Strand Buffer SB 15 uL DNA Polymerase DP 5 ul RNaseH RH 1 uL Total 130 pL 3 Add 130 pL of the Second Strand Master Mix to each well with sample in it and mix gently by pipetting up and down several times Cover the plate with a sealing film 4 Incubate the 96 well plate at 16 C for 2 hours If using a thermal cycler pre cool to 16 C before starting the reaction 5 Stop the reaction by placing the 96 well plate on ice 6 Immediately proceed to purification of the double stranded cDNA template CDNA PURIFICATION In order to proceed to the RNA Transcript Labeling step purification of the double stranded cDNA template is necessary Use of unpuri fied cDNA has not been validated with the biotin labeling system provided in this kit A suggested protocol for cDNA purification is outlined in Appendix C D RNA TRANSCRIPT LABELING 1 Thaw vials RB B and D Mix well gently centrifuge and keep on i
14. ity and quality of the resulting cDNA Starting with total cellular RNA minimizes loss of low abundance transcripts during mRNA isolation Most commercially available RNA isolation reagents are compatible with the 100 Reaction Single Round RNA Amplification and Label ing System Total RNA samples should be in water or buffer free of protein DNA cellular material organic solvents salts and other RNA isolation reagents RNA purity can be assessed via a spectrophotometer with accept able Azgo Azgo ratios between 1 8 and 2 1 RNA integrity can be evaluated by analysis on a bioanalyzer RIN values should be between 7 and 10 Alternatively the presence of discrete bands at 28S and 18S rRNA at an abundance ratio of 2 1 ratio 28S 18S in denaturing agarose gel electrophoresis indicates intact RNA Both the input RNA and amplified aRNA should be used immedi ately after purification or stored at lt 70 C until use Results may vary for samples subject to multiple freeze thaw events A FiRST STAND SYNTHESIS 1 Thaw vials dN P FB D and W Gently mix centrifuge 5 sec and place on ice until needed Briefly centrifuge all tubes contain ing enzymes immediately prior to use Place 250 ng to 5 ug of total RNA into the wells of a PCR plate Then add 1 uL Promoter Primer P to each well Add an appropriate volume of Nuclease free Water W to the RNA Primer mix so that the total volume in each well is 13 pL Cover the 96
15. ted and purified RNA e 0 2 ml 0 6 ml and 1 5 ml microcentrifuge tubes e Sterile aerosol barrier nuclease free pipette tips e RNase decontamination solution e Molecular Biology Grade Ethanol 100 Methods and Procedures NOTE PLEASE READ THE ENTIRE PROCEDURE BEFORE STARTING Upon thawing of solutions gently hand mix or vortex the reagents prior to use to ensure a homogenous solution The entire process involves four major steps that are accomplished by using the 100 Reaction Single Round Amplification and Biotin Labeling System with recommended purification and analysis procedures With the exception of all purifications the entire procedure may be performed in a thermal cycler see Table 3 below To avoid condensa tion set the thermal cycler lid temperature to 5 C above the reaction temperature Alternatively heating blocks with heated lids water baths or hybridization ovens set at the prescribed temperatures can be used For the sake of consistency of common reaction components we recom mend making master mixes for each step of the procedure when perform ing more than one reaction The reaction tables show exact reactant vol umes per reaction While making a master mix Include 1096 extra volume of each common reaction component in the master mix to make up for losses in distributing into each reaction vial Table 3 Thermal Cycler Amplification Program Process C Time 70 10 minutes Denaturation 42 3 minutes
16. well plate with a sealing film Incubate the 96 well plate for 10 minutes at 70 C to denature the RNA NOTE f there is condensation on the seal cover after the incubation centrifuge briefly to collect samples at the bottom of the wells Keep the multi well plate at 4 C until addition of the First Strand Master Mix in step A6 While denaturing prepare the First Strand Master Mix by combining the reagents Table 4 in a sterile nuclease free microcentrifuge tube Assemble enough master mix for all sam ples plus 10 overage to accommodate pipeting error Gently mix the reagents by pipetting up and down several times centri fuge and place the First Strand Master Mix on ice Table 4 First Strand Master Mix Component Vial ID Vol per Reaction First Strand Buffer FB 2 0 uL DTT D 2 0 uL dNTP Mix dN 1 0 uL Reverse Transcriptase RT 1 0 uL RNase Inhibitor I 1 0 uL Total 7 0 pL While the 96 well plate is incubating at 4 C add 7 uL of First Strand Master Mix to each well and mix by pipeting up and down several times Cover the 96 well plate with a sealing film Incubate the 96 plate at 42 C for 2 hours NOTE f there is condensation on the seal cover after the incubation centrifuge briefly to collect samples at the bottom of the wells Stop the reaction by placing the 96 well plate on ice B SECOND STRAND CDNA SYNTHESIS 1 Thaw vials dN SB and W mix briefly centrifuge and keep on ice 2 S
17. x C
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