SuperScript RNA Amplification System
Contents
1. 175 Fluorescence 14 19 24 29 34 39 44 49 54 59 64 69 7a 79 84 Time seconds RNA 6000 Ladder from Ambion for use with the RNA 6000 LabChip kits 175 E 4 IS 15 0 1 0 F L 125 5 10 0 t 20 40 B75 1 O2 4 6 0 kb 50 L L i v LN l 0 14 19 24 29 34 39 44 49 54 59 64 69 74 79 84 Time seconds 15 Dye Coupling to Amino Allyl aRNA Introduction Note Before Starting Coupling Buffer Dye Information Important 16 This section provides a general protocol for labeling amino allyl aRNA with Cy3 or Cy5 monofunctional NHS reactive fluorescent dyes You must first calculate the yield of amino allyl aRNA as described on page 14 before proceeding to dye coupling The following items will be used in the procedure e Speed vac e Sodium tetraborate decahydrate 500 g Fisher cat no S248 500 e HCl e 0 22 micron syringe filter or vacuum flask filter s DMSO e DEPC treated water e Fluorescent dye see above e Purification components listed on page 12 To prepare the Coupling Buffer used in the labeling reaction 1 Dissolve 3 8 g of sodium tetraborate decahydrate in 100 ml of DEPC treated water 2 Adjust the pH with HCI to 8 5 3 Sterilize with a 0 22 micron syringe filter for individual reaction volumes or vacuum flask filter This kit has been validated with the following dyes and dye packs CyDye P2. non exclusive non transferable right without the right to resell repackage or further sublicense under these patents to use this product for research and development purposes No other license is granted to the buyer whether expressly by implication by estoppel or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially such as the generation of commercial databases or the provision of clinical diagnostics This product is sold pursuant to authorization from Incyte Corporation and Incyte Corporation reserves all other rights under these patents For information on purchasing a license for purposes other than research and develop ment please contact Incyte Corporation Corporate Licensing 3160 Porter Drive Palo Alto CA 94304 Phone 650 843 7313 Fax 650 845 4696 CyDye Cy3 and Cy5 are trademarks of Amersham Biosciences Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are
3. RPN 5661 Add 5 ul DMSO directly to each dye vial Large size PA 23001 and PA 25001 Add 45 ul DMSO directly to each dye vial Use 5 ul of this DMSO dye solution in the next step Add 5 ul of the DMSO dye solution to the tube from Step 3 above 6 Mix well and incubate the tube at room temperature in the dark for 30 45 minutes Store any unused dye solution according to manufacturer s directions 7 Purify the dye coupled amino allyl aRNA as described in aRNA Purification page 12 Be careful to minimize exposure of the reaction to light We recommend collecting the purified dye labeled sample in an RNase free amber collection tube To calculate the amount of coupled dye see below Calculating the The following table shows the absorbance and baseline wavelengths for Cy3 Amount of and Cy5 dyes Coupled Dye Dye Absorbance Wavelength Baseline Wavelength Cy3 550 nm 650 nm Cy5 650 nm 750 nm To calculate the amount of coupled dye 1 Transfer the undiluted purified dye coupled amino allyl aRNA from Step 11 page 12 into a clean cuvette and scan at 240 800 nm using a UV Visible spectrophotometer If you are using a 100 pl cuvette transfer the entire sample for smaller cuvettes transfer an appropriate amount of sample Note The labeled aRNA must be purified as described on page 12 before scanning as any unreacted dye will interfere with the detection of labeled aRNA 2 Calculate the amount of fluorescently la
4. cDNA Note For multiple reactions prepare a master mix with a 5 10 overage to enable accurate pipetting Note For optimal results reagents should be ice cold when they are added to the reaction 1 The double stranded cDNA can be stored at 20 C until you are ready to perform the rest of the procedure Proceed to cDNA Purification on the Add the following components to the reaction tube from step 6 previous page on ice Component Sample DEPC treated water 91 ul 5X Second Strand Buffer 30 pl 10 mM dNTP Mix 3 ul DNA Polymerase I 10 units pl 4 ul DNA Ligase 10 units l 1 pl RNase H 2 units Iu 1ul Total Volume 150 ul Mix the contents gently by pipetting up and down Incubate the reaction mixture at 16 C for 2 hours After incubation place the tubes on ice following page cDNA Purification Introduction Before Starting Purification Procedure In this step you purify the double stranded cDNA using the spin columns provided in the kit The following items are supplied by the user e Microcentrifuge e Speed vac The following items are supplied in the kit e Spin Cartridges pre inserted into collection tubes e Recovery Tubes e cDNA Loading Buffer plus isopropanol see page vi for preparation e cDNA Wash Buffer plus ethanol see page vi for preparation e DEPC treated water Use the following procedure to purify the cDNA Note Before starting the procedure be sure to add iso
5. listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Limited Warranty MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless al
6. to enable accurate pipetting Note If you are setting up a control reaction recommended for first time users use 1 ul of the Control HeLa RNA supplied in the kit 500 ng ul 1 6 Mix and briefly centrifuge each component before use In a 1 5 ml RNase free tube add the following Component Sample 100 5000 ng of total RNA or 5 250 ng of mRNA lt 9 pl T7 Oligo dT Primer 1 pl DEPC treated water to 10 pl For the control reaction use 1 ul of the supplied Control HeLa RNA 500 ng ul Incubate the tube at 70 C for 10 minutes and then place on ice for at least 1 minute Centrifuge the tube briefly to collect the contents and add the following to the tube at room temperature Component Volume 5X First Strand buffer 4 ul 0 1 M DTT 2 ul 10 mM dNTP Mix 1 pl RNaseOUT 40 U pl 1 u SuperScript III RT 200 U nl 2ul Total Reaction Volume 20 pl Mix gently and then centrifuge the tube briefly to collect the contents Incubate the tube at 46 C for 2 hours Incubate the tube at 70 C for 10 minutes to inactivate the reverse transcriptase Centrifuge the tube briefly to collect the contents and place the tube on ice After incubation proceed immediately to Second Strand cDNA Synthesis next page Continued on the next page cDNA Synthesis continued Second Strand cDNA Synthesis After first strand synthesis immediately perform the following second strand synthesis reaction to generate double stranded
7. 6 16 hours Optional Add 2 ul of DNase I to the tube Gently mix and centrifuge briefly to collect the contents of the tube and then incubate at 37 C for 30 minutes Proceed to aRNA Purification page 12 11 aRNA Purification Introduction Before Starting Purification Procedure 12 In this step you purify the aRNA using the spin columns provided in the kit The following items are supplied by the user e Microcentrifuge e Speed vac e 100 ethanol The following items are supplied in the kit e Spin Cartridges pre inserted into collection tubes e Recovery Tubes e aRNA Binding Buffer e aRNA Wash Buffer plus ethanol see page vii for preparation e DEPC treated water Use the following procedure to purify the aRNA Note Before starting the procedure be sure to add ethanol to the aRNA Wash Buffer supplied in the kit as described on page vii 1 Add 160 ul of aRNA Binding Buffer to the reaction tube from Step 6 on page 11 or 11 The total volume should be 200 pl Mix thoroughly by pipetting up and down 2 Add 100 pl of 100 ethanol to the reaction tube Mix thoroughly by pipetting up and down 3 Each Spin Cartridge is pre inserted into a collection tube Load the entire aRNA buffer solution directly onto the Spin Cartridge 4 Centrifuge at 12 000 x g in a microcentrifuge for 15 seconds at room temperature Remove the collection tube and discard the flow through 5 Place the Spin Cartridge in the same c
8. 7 promoter Following second strand synthesis and purification the cDNA template is amplified via in vitro transcription using T7 RNA polymerase in an optimized enzyme and buffer formulation This step transcribes antisense RNA aRNA molecules complementary to the original mRNA targets The amplified aRNA is then ready to use in applications such as gene expression profiling Amplified RNA is ideal for gene expression profiling from very small amounts of starting material because it preserves the relative abundance of the different mRNA sequences in the original sample allowing you to compare relative quantities across experiments This system has been optimized for use with 100 5000 ng of total RNA or 5 250 ng of poly A RNA as starting material The amplified unlabeled RNA can be subsequently labeled by reverse transcription Alternatively amino allyl modified nucleotides can be incorporated into the RNA in the in vitro transcription reaction for subsequent labeling and detection 5S AMMA 3 mRNA TTTTT T7 T7 Oligo dT First strand cDNA synthesis Se TTTTT T7 cDNA Second strand cDNA synthesis and purification AAAAA T7 Double stranded sa TN TTTT T7 cDNA In vitro transcription and purification Ne UU SO UUUUU Amplified aRNA Ne UU Continued on next page Overview continued Advantages of the System SuperScript Ill Reverse Transcriptase T7 Oligo dT Control RNA e Optimized reagent
9. aRNA Wash Buffer you must add 100 ethanol to create the final buffer see below DEPC treated Water Spin Cartridges Recovery Tubes The cDNA Loading Buffer must be mixed with 100 isopropanol prior to use The Loading Buffer plus isopropanol is stable for at least six months at room temperature Add the amount of isopropanol indicated below directly to the bottle of Loading Buffer Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the isopropanol Component Amount cDNA Loading Buffer 9 ml entire bottle 100 Isopropanol 3 ml Final Volume 12 ml The cDNA Wash Buffer must be mixed with 100 ethanol prior to use The cDNA Wash Buffer plus ethanol is stable for at least six months at room temperature Add the amount of ethanol indicated below directly to the bottle of cDNA Wash Buffer Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the ethanol Amount cDNA Wash Buffer 4 ml entire bottle 100 Ethanol 12 ml Final Volume 16 ml Continued on next page Kit Contents and Storage continued Preparing aRNA The aRNA Wash Buffer must be mixed with 100 ethanol prior to use The Wash Buffer with aRNA Wash Buffer plus ethanol is stable for at least six months at room Ethanol temperature Add the amount of ethanol indicated below directly to each bottle of aRNA Wash Buffer Be sure to mark the appropriate checkbox on the bottle to indicate that you have add
10. ase free microcentrifuge tubes are available from several suppliers If it is necessary to decontaminate untreated tubes soak the tubes overnight in a 0 01 v v aqueous solution of diethylpyrocarbonate DEPC rinse the tubes with sterile distilled water and autoclave the tubes TM You can use RNase Away Reagent a non toxic solution available from Invitrogen see page 19 to remove RNase contamination from surfaces For further information on controlling RNase contamination see Ausubel et al 1994 and Sambrook et al 1989 Amount of RNA This system is optimized for use with 100 5000 ng of total RNA or 5 250 ng of purified poly A RNA Larger amounts of starting material may lead toa decrease in amplification efficiency while smaller amounts may result in a decrease in amplification specificity Continued on next page Isolating RNA continued Isolating RNA Checking the RNA Quality Storing RNA To isolate total RNA we recommend the PureLink Micro to Midi Total RNA Purification System TRIzol Reagent or for high throughput applications the PureLink 96 RNA Purification System To isolate mRNA we recommend the FastTrack 2 0 mRNA Isolation Kits or the FastTrack MAG mRNA Isolation Kits Ordering information is provided on page 19 After you have isolated the RNA check the quality of your RNA preparation as described on the following page We recommend checking the quality of the RNA preparation using t
11. at low to medium heat until the sample volume is reduced to lt 20 pl Be careful not to overdry the sample as this may result in sample loss Proceed to In Vitro Transcription next page In Vitro Transcription Introduction Before Starting Note In Vitro Transcription Unlabeled aRNA 10 In this step you generate aRNA from the double stranded cDNA using T7 RNA Polymerase An alternative protocol is provided for generating aRNA with amino allyl UTP for subsequent indirect labeling with fluorescent dyes The following items are supplied by the user e Microcentrifuge e Air incubator or thermal cycler set at 37 C heat block is not recommended e Optional 50 mM amino allyl UTP The following items are supplied in the kit e DEPC treated water e T7 Enzyme Mix s 10X T7 Reaction Buffer e DNase I optional e 100mM ATP s 100mM CTP s 100mM GTP s 100mM UTP The yield of aRNA will increase with longer in vitro transcription incubation times up to 16 hours For maximum yield an incubation time of at least 12 hours is recommended Use the following procedure to generate unlabeled aRNA See the following page for a procedure to generate amino allyl aRNA For multiple reactions prepare a master mix with a 5 10 overage to enable accurate pipetting 1 Add DEPC treated water to the tube containing purified cDNA from Step 11 page 9 to bring the total volume to 23 ul 2 Add the following to the tube at room te
12. beled aRNA using a formula below Cy3 pmole Asso As50 0 15 x 100 elution volume Cy5 G pmole Asso A750 0 25 x 100 elution volume 17 Troubleshooting 28S and 18S bands are not observed after isolation of total RNA and agarose gel electrophoresis 28S band is diminished or low molecular weight RNA appears in the gel Yield of aRNA from the control reaction is low 18 Too little RNA loaded To analyze total RNA by gel electrophoresis you need on the gel at least 250 ng of RNA RNA is degraded due Follow the guidelines on page 4 to avoid RNase to RNase activity contamination Use a fresh sample for RNA isolation RNA is degraded Follow the guidelines on page 4 to avoid RNase contamination Use a fresh sample for RNA isolation Incubation temperatures were incorrect Check the incubation temperatures of all the reactions Incorrect reaction conditions used Verify that all reaction components are included in the reaction and use reagents provided in the system Verify the reaction conditions using the Control HeLa RNA provided in the kit If condensation forms inside the tubes during incubation spin the tube briefly to remix the components and perform the reaction in a different incubator air incubation is recommended Condensation formed in the reaction tubes Poor quality RNA used or RNA is degraded RNase contamination Use the RNaseOUT included in the kit to prevent RNA de
13. can use the Quant iT RNA Assay Kit from Molecular Probes Cat no Q 33140 or the RiboGreen RNA Quantitation Kit from Molecular Probes Cat no R 11490 for highly sensitive quantitation of small amounts of RNA using a fluorescence microplate reader See the product information sheet for each kit for detailed protocols Use 1 pl of the purified aRNA from Step 11 page 12 in the quantitation reaction The following general protocol may be used to calculate the yield of the aRNA using A2 absorbance 1 Aliquot 1 pl of the purified aRNA from Step 11 page 12 into a clean cuvette and dilute it 1 10 to 1 100 using DEPC treated water As a general guideline begin by diluting the aRNA sample 1 10 if you used 100 ng of total RNA starting material and 1 100 if you used 500 ng total RNA starting material 2 Scan the sample at 260 nm using a UV Visible spectrophotometer Be sure to blank the spectrophotometer using DEPC treated water before the reading Note The Az reading should fall within the standard specification for the spectrophotometer typically 0 1 1 0 OD If it falls outside this range adjust the dilution and re scan If the A2 reading is too low use a lower dilution if it s too high use a higher dilution 3 Transfer the sample back into the Recovery Tube for storage 4 Calculate the yield of aRNA using the formula below Total aRNA yield g ml Az x 40 pg ml RNA x dilution factor For example if you diluted 1 ul o
14. e store the RNA at 80 C cDNA Synthesis Introduction After you have isolated RNA and checked the quality of your RNA preparation you are ready to synthesize double stranded cDNA Before Starting The following items are supplied by the user e 100 5000 ng of total RNA or 5 250 ng of poly A RNA e Vortex mixer e Incubator or thermal cycler set at 46 C and 70 C e Refrigerated water bath set at 16 C s 1 5 ml RNase free microcentrifuge tubes e Ice The following items are supplied in the kit e T7 Oligo dT primer e 10mM dNTP Mix e 5X First Strand Buffer s 0 1MDITT e RNaseOQUT e SuperScript II RT e DNA Polymerase I e DNA Ligase e 5X Second Strand Buffer e DEPC treated water e RNase H e Control HeLa RNA optional see page 2 Note For optimal results the reagents used in second strand cDNA synthesis should be ice cold when they are added to the reaction tube RNaseOUT RNaseOUT Recombinant RNase Inhibitor has been included in the system to Recombinant safeguard against degradation of target RNA due to ribonuclease RNase Inhibitor contamination of the RNA preparation Continued on next page cDNA Synthesis continued First Strand cDNA The following procedure is designed to convert 100 5000 ng of total RNA or 5 250 ng of purified poly A RNA into first strand cDNA Synthesis Note The following procedure is for a single reaction For multiple reactions prepare a master mix with a 5 10 overage
15. ed the ethanol Amount aRNA Wash Buffer 7 ml entire bottle 100 Ethanol 21 ml Final Volume 28 ml vii Accessory Products Additional The following related products are available separately from Invitrogen To Products order visit www invitrogen com or contact Technical Support see page 21 SuperScript Indirect RNA Amplification System L1016 02 RNase Away Reagent 250 ml 10328 011 PureLink Micro to Midi Total RNA Purification 12183 018 System PureLink 96 RNA Purification System 12173 011 TRIzol Reagent 100 ml 15596 026 200 ml 15596 018 FastTrack 2 0 mRNA Isolation Kit 6reactions K1593 02 18 reactions K1593 03 Quant iT RNA Assay Kit 1000 reactions Q 33140 RiboGreen RNA Quantitation Kit 200 2000 R 11490 reactions RNaseOUT Recombinant Ribonuclease Inhibitor 5000 units 10777 019 1 2 E Gel Starter Pack 6gelsand G6000 01 base UltraPure DEPC treated water 4x1 25ml 10813 012 viii Overview Introduction Experimental Outline TM The SuperScript RNA Amplification System is a highly robust and efficient system for amplifying mRNA from small starting quantities of total RNA or purified poly A RNA This kit is based on the isothermal RNA amplification protocol developed in the laboratory of Dr James Eberwine Van Gelder et al 1990 It uses SuperScript III Reverse Transcriptase to synthesize first strand cDNA primed with an anchored oligo dT primer containing a T
16. f a 100 ul volume of aRNA at 1 50 and the A2 is 0 5 then 0 5 x 40 ug ml RNA x 50 1000 pg ml In a 100 ul volume you would have 100 pg of aRNA The expected yield using the Control HeLa RNA 500 ng provided in the kit depends on whether the aRNA is labeled or unlabeled as well as the length of the in vitro transcription reaction If you do not achieve the minimum yields specified below for the control reaction see Troubleshooting on page 16 Note The expected yields listed below assume a 14 hour in vitro transcription reaction Type of aRNA Expected Yield Unlabeled gt 35 ug Amino allyl labeled gt 20 pg Determining aRNA Quality Determining aRNA Quality Using the Agilent 2100 Bioanalyzer You can use the Agilent 2100 bioanalyzer with the RNA 6000 LabChip Kit to analyze you aRNA sample We do not recommend using the bioanalyzer to determine aRNA quantity see the previous page for recommended quantitation methods To analyze samples using the bioanalyzer use 1 ul of the purified aRNA from Step 11 page 12 The bioanalyzer graph for a typical amplification reaction will shown a population of aRNAs ranging from 0 2 kb to 4 kb with a peak between 1 kb and 2 kb average aRNA size gt 1 kb Examples are shown below and an RNA ladder is provided for size comparison Unlabeled aRNA 154 Fluorescence 3 14 19 24 29 34 39 44 49 54 59 64 69 74 79 84 Time seconds Amino allyl labeled aRNA 20 0 F T
17. gradation Check the quality of your RNA preparation see page 5 If RNA is degraded use fresh RNA RT inhibitors are present in your RNA sample Inhibitors of RT include SDS EDTA guanidinium chloride formamide sodium phosphate and spermidine Gerard 1994 Test for the presence of inhibitors by mixing 1 ug of Control HeLa RNA with 25 ug total RNA or 1 ug mRNA and compare the yields of aRNA amplification Improper storage of Store the enzyme at 20 C SuperScript III RT Reagents were not Repeat the procedure being careful to briefly vortex properly mixed before and centrifuge each reagent before first strand cDNA first strand synthesis synthesis Continued on next page Troubleshooting continued Average aRNA size is Incubation Check the incubation temperatures of all the reactions lt 500 nucleotides for temperatures were both sample RNA and_ incorrect control RNA reactions RNase contamination Use the RNaseOUT included in the kit to prevent RNA degradation Problem with gel Improperly formulated agarose gels may provide analysis of aRNA inaccurate estimates of aRNA size Analyze an RNA ladder in an adjacent lane to confirm the size of the aRNA products Average aRNA size is Poor quality RNA Check the quality of your RNA preparation see lt 500 nucleotides for used or sample RNA page 5 If RNA is degraded use fresh RNA sample RNA but is gt 1 is degraded kb for control RNA Ineffic
18. he Agilent 2100 bioanalyzer and an RNA 6000 LabChip Kit which is ideal for analyzing small quantities of RNA You may also use agarose ethidium bromide gel electrophoresis for larger quantities The Agilent 2100 bioanalyzer and RNA 6000 LabChip Kit are suitable for analyzing very small quantities of RNA as low as 200 pg In the bioanalyzer graph for total human RNA the 28S rRNA peak should be approximately twice the size of the 18S rRNA peak If the peaks appear similar in size or if additional peaks appear on the graph the RNA may be degraded Agarose Gel Electrophoresis is suitable for analyzing larger amounts of RNA gt 500 ng total RNA You can use a 1 agarose gel or a denaturing agarose gel Ausubel et al 1994 For total human RNA using a regular agarose gel mRNA will appear as a smear from 0 5 to 9 kb and 28S and 18S rRNA will appear as bands at 4 5 kb and 1 9 kb respectively The 28S band should be twice the intensity of the 18S band If you are using a denaturing gel the rRNA bands should be very clear and sharp If you do not load enough RNA the 285 band may appear to be diffuse A smear of RNA or a lower intensity 28S band with an accumulation of low molecular weight RNA on the gel are indications that the RNA may be degraded If you have problems with RNA quality refer to Troubleshooting on page 16 After preparing the RNA we recommend that you proceed directly to First Strand cDNA Synthesis on page 6 Otherwis
19. ient labeling Follow all purification steps carefully and without due to improper modification purification Starting amount of Increase the amount of starting RNA RNA is too low reaction 19 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 199 Eberwine Technology Trademarks 20 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or
20. invitrogen SuperScript RNA Amplification System For generating amplified mRNA from small starting quantities of RNA Catalog nos L1016 01 Version E 7 June 2010 25 0740 ii Table of Contents Kit Contents and Storage nn ansannseneenesahnneslan nn BD DER ee Y Accessory S LT Viii OVERVIEW A a ee Eileen E E E T E EN 1 LWS Us L 4 Isolating RNAus une ae a Rinne Hin Rep a a RaRa EK EAEE Eara 4 DNA SyAthesis ann 22 2 eae o a aE tisha aaR a Eee Heche eee ds eta loaae baleen taba Uti eas 6 EDNA Putificationsa rare area dita cenitne A R e 9 In Viro Transcription zu 522 22 ar EEE asien EI EHEN Beane eee 10 aRNA Purification snr ker nee ame De san 12 ADD avd SID ira oR B Br Me Bi No Sp rra NENV a HNS N hna MeN OE HPS ane 14 Determining aRNA Yield T TTT T 14 Determining aRNA Quality TTT 15 Dye Coupling E He T E O PTT 16 Troubleshooting ists a ui eile ee nalen leise fae nae ee Ata 18 Purchaser Notification ussere bet sea 20 T amp chnical Suppott nennsiineeskenei ss Rit ensehtaheikelikngehenlegeiieientesgehstle 21 References 0 a ee esse 22 iii iv Kit Contents and Storage TM Shipping and The SuperScript RNA Amplification System is shipped in two modules The Storage Core Module is shipped on dry ice while the Purification Module is shipped at room temperature Upon receipt store the components of the Core Module at 20 C and store the components of the Purification Module at room temperature Co
21. itrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 22 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
22. its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com This product may be covered by one or more of U S Patent numbers 5 545 522 5 514 545 and 5 958 688 licensed exclusively to Incyte Corporation The purchase of this product conveys to the buyer whether employed in academia government not for profit entity or for profit entity the limited
23. l product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 21 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Baugh L R Hill A A Brown E L and Hunter C P 2001 Quantitative analysis of mRNA amplification by in vitro transcription Nucleic Acids Res 29 E29 Chirgwin J M Przybyla A E MacDonald R J and Rutter W Z 1979 Isolation of Biologically Active Ribonucleic Acid from Source
24. mine the efficiency of the amplification procedure We recommend that you perform the complete procedure using the control HeLa RNA if you are a first time user of the system Methods for determining the aRNA yield and quality from the control HeLa RNA are provided on pages 14 15 Continued on next page Overview continued Materials Supplied by the User Product Qualification In addition to the kit components you should have the following items on hand before using this kit 100 5000 ng of total RNA or 5 250 ng of poly A RNA Note that this kit has been optimized for use with total RNA and purification of poly A RNA is not required in most cases Amino allyl modified nucleotides optional for use in the in vitro transcription reaction Vortex mixer Microcentrifuge Speed vac concentrator Spectrophotometer Recommended Agilent 2100 bioanalyzer and RNA 6000 LabChip Kit for analyzing starting material and final aRNA product Optional Denaturing agarose gel for analyzing starting material and 1 2 agarose gel for analyzing final aRNA product Aerosol resistant pipette tips Air incubator or thermal cycler Refrigerated water bath 1 5 ml RNase free microcentrifuge tubes Ice 100 Isopropanol 100 Ethanol This kit was verified using 500 ng of total HeLa RNA in a standard amplification reaction as described in this manual After purification the amount of aRNA was calculated using A2 absorbance as described on
25. mperature Component Volume 100 mM ATP 1 5 ul 100 mM CTP 1 5 ul 100 mM GTP 1 5 ul 100 mM UTP 1 5 ul 10X T7 Reaction Buffer 4ul T7 Enzyme Mix 7 ul Total Reaction Volume 40 ul Protocol continued on next page Continued on next page In Vitro Transcription continued In Vitro Transcription Unlabeled aRNA continued In Vitro Transcription Amino Allyl aRNA Protocol continued from previous page 3 4 5 Gently mix by hand and centrifuge briefly to collect the contents of the tube Incubate at 37 C for 6 16 hours Optional Add 2 ul of DNase I to the tube Gently mix and centrifuge briefly to collect the contents of the tube and then incubate at 37 C for 30 minutes Proceed to aRNA Purification page 12 Use the following procedure to generate aRNA with amino allyl UTP for subsequent labeling with fluorescent dye and array hybridization For multiple reactions prepare a master mix with a 5 10 overage to enable accurate pipetting 1 Add DEPC treated water to the tube containing purified cDNA from Step 11 page 9 to bring the total volume to 22 ul Add the following to the tube at room temperature Component Volume 100 mM ATP 1 5 pl 100 mM CTP 1 5 pl 100 mM GTP 1 5 pl 100 mM UTP 0 75 ul 10X T7 Reaction Buffer 4ul T7 Enzyme Mix 7 pl Amino allyl UTP 50 mM 1 75 ul Total Reaction Volume 40 ul Gently mix and centrifuge briefly to collect the contents of the tube Incubate the tube at 37 C for
26. ollection tube and add 500 pl of aRNA Wash Buffer prepared as described on page vii to the column 6 Centrifuge at 12 000 x g for 15 seconds at room temperature Remove the collection tube and discard the flow through Repeat Steps 5 6 Place the Spin Cartridge in the same collection tube and centrifuge at full speed for an additional 2 minutes to dry the column Remove the collection tube and discard the flow through Procedure continued on next page Continued on next page aRNA Purification continued Purification Procedure continued Procedure continued from previous page 9 Place the Spin Cartridge into a new Recovery Tube supplied in the kit Note For fluorescent labeled aRNA we recommend using an amber recovery tube not supplied to avoid photobleaching 10 Add 100 ul of DEPC treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute 11 Centrifuge at 12 000 x g for 2 minutes at room temperature to collect your purified aRNA The eluate contains your purified aRNA To calculate the yield of aRNA proceed to Determining aRNA Yield on page 14 To label amino allyl aRNA with fluorescent dye proceed to Dye Coupling to Amino Allyl aRNA page 16 Stored the sample at 80 C 13 Appendix Determining aRNA Yield Determining aRNA Yield Using an RNA Quantitation Kit Determining aRNA Yield Using Aaso Absorbance Expected Yield for Control HeLa RNA 14 You
27. ost Labeling Reactive Dye Pack 12 vials each Cy3 and Cy5 Amersham Biosciences RPN 5661 Cy3 Mono Reactive Dye Pack Amersham Biosciences PA 23001 Cy5 Mono Reactive Dye Pack Amersham Biosciences PA 25001 Fluorescent dyes are sensitive to photobleaching When preparing the reaction be careful to minimize exposure of the dye solution to light The dye coupling reaction must be incubated in the dark DMSO is hygroscopic and will absorb moisture from the air Water absorbed from the air will react with the NHS ester of the dye and significantly reduce the coupling reaction efficiency Keep the DMSO supplied in the kit in an amber screw capped vial at 20 C and let the vial warm to room temperature before opening to prevent condensation Continued on next page Dye Coupling to Amino Allyl aRNA continued Coupling Reaction Follow the steps below to couple fluorescent dye to your amino allyl aRNA 1 Calculate the yield of amino allyl aRNA as described on page 14 Determine the volume of purified sample that contains 5 pg of amino allyl aRNA 2 Aliquot the volume containing 5 pg of amino allyl aRNA into a 1 5 ml RNase free microcentrifuge tube and place in a speed vac Evaporate at low heat until the sample volume is reduced to lt 1 pl Be careful not to overdry 3 Add 5 ul of Coupling Buffer to the tube see recipe on previous page TM 4 Prepare the Cy3 or Cy5 dye as follows Individual reaction size
28. page 14 The quality of the aRNA was verified using agarose gel electrophoresis The representation of a group of housekeeping genes of various expression levels was confirmed using the aRNA in quantitative RT PCR with Certified LUX Primer Sets Methods Isolating RNA Introduction High quality intact RNA is essential for full length high quality cDNA synthesis In this step you isolate total RNA or poly A RNA using a method of choice Note This kit has been optimized for use with total RNA Purification of poly A RNA is not required in most cases The quality of the RNA is critical for RNA amplification In labeling and array Important hybridization applications the presence of contaminants in the RNA may significantly increase background fluorescence in the microarrays Carefully follow the recommendations below to prevent contamination General Handling When working with RNA of RNA e Use disposable individually wrapped sterile plasticware e Use aerosol resistant pipette tips for all procedures e Use only sterile new pipette tips and microcentrifuge tubes e Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin e Use proper microbiological aseptic technique when working with RNA e Dedicate a separate set of pipettes buffers and enzymes for RNA work e Microcentrifuge tubes can be taken from an unopened box autoclaved and used for all RNA work RN
29. propanol to the cDNA Loading Buffer supplied in the kit and ethanol to the cDNA Wash Buffer supplied in the kit as described on page vi 1 Add 500 ul of cDNA Loading Buffer prepared as described on page vi to the reaction tube from Second Strand cDNA Synthesis Step 3 page 8 The total volume in the tube should be 650 pl Mix thoroughly by pipetting up and down 2 Each Spin Cartridge is pre inserted into a collection tube Load the cDNA buffer solution directly onto the Spin Cartridge 4 Centrifuge at 12 000 x g at room temperature in a microcentrifuge for 1 minute Remove the collection tube and discard the flow through 5 Place the Spin Cartridge in the same collection tube and add 700 ul of CDNA Wash Buffer prepared as described on page vi to the column 6 Centrifuge at 12 000 x g at room temperature for 2 minutes Remove the collection tube and discard the flow through 7 Place the Spin Cartridge in the same collection tube and centrifuge at 12 000 x g at room temperature for an additional 4 minutes Remove the collection tube and discard the flow through 8 Place the Spin Cartridge into a new Recovery Tube supplied in the kit 9 Add 100 pl of DEPC treated water to the center of the Spin Cartridge and incubate at room temperature for 2 minutes 10 Centrifuge at 12 000 x g at room temperature for 1 minutes to collect your purified cDNA The eluate contains your purified cDNA 11 Place the eluate in a speed vac and evaporate
30. re Module The Core Module includes enough reagents for 20 reactions The components of the Core Module should be stored at 20 C Amount SuperScript III 200 U pl 40 ul Reverse Transcriptase 5X First Strand Buffer 250 mM Tris HCl pH 8 3 room temp 90 ul 375 mM KCI 15 mM MgCl Dithiothreitol DTT 0 1 M DTT in water 50 pl 10 mM dNTP Mix dATP dGTP dCTP and dTTP in DEPC 80 ul treated water T7 Oligo dT primer In DEPC treated water 20 pl RNaseOUT 40 U ul 30 pl Recombinant Ribonuclease Inhibitor Control HeLa RNA 500 ng pl in HE buffer 20 ug Polymerase Bl 25 wl 100 mM Tris HCl pH 6 9 450 mM KCl 700 pl Reaction Buffer 23 mM MgCl 50 mM NH SO stabilizers T7 Enzyme Mix Includes T7 RNA Polymerase proprietary 140 pl formulation 10X T7 Reaction Proprietary formulation 80 pl Buffer Td T 305 d 30 Continued on next page N B Kit Contents and Storage continued Purification Module Preparing cDNA Loading Buffer with Isopropanol Preparing cDNA Wash Buffer with Ethanol vi The Purification Module includes enough reagents and columns for 20 reactions The components of the Purification Module should be stored at room temperature cDNA Loading Buffer you must add 100 isopropanol to create 9 ml the final buffer see below cDNA Wash Buffer you must add 100 ethanol to create the 4 ml final buffer see below aRNA Binding Buffer no additional preparation is necessary
31. s Enriched in Ribonucleases Biochem 18 5294 5299 Chomczynski P and Sacchi N 1987 Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate Phenol Chloroform Extraction Anal Biochem 162 156 159 Nygaard V Loland A Holden M Langaas M Rue H Liu F Myklebost O Fodstad O Hovig E and Smith Sorensen B 2003 Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance BMC Genomics 4 11 Puskas L G Zvara A Hackler L Jr and Van Hummelen P 2002 RNA amplification results in reproducible microarray data with slight ratio bias Biotechniques 32 1330 4 1336 1338 1340 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Van Gelder R N von Zastrow M E Yool A Dement W C Barchas J D and Eberwine J H 1990 Amplified RNA synthesized from limited quantities of heterogeneous cDNA Proc Natl Acad Sci USA 87 1663 7 Wang J Hu L Hamilton S R Coombes K R and Zhang W 2003 RNA amplification strategies for cDNA microarray experiments Biotechniques 34 394 400 Related Articles Links Zhao H Hastie T Whitfield M L Borresen Dale A L and Jeffrey S S 2002 Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis BMC Genomics 3 31 2004 2006 Inv
32. s and protocol ensure highly robust and reproducible reactions e SuperScript III Reverse Transcriptase in the first strand synthesis reaction produces higher yields of CDNA and more complete representation of the mRNA population from very small amounts of starting material e System generates aRNA with a greater average length than comparable kits e System includes all major reagents and materials for preparing amplified RNA TM SuperScript III Reverse Transcriptase is an engineered version of M MLV RT with reduced RNase H activity and increased thermal stability The enzyme can be used to synthesize first strand cDNA from total RNA or mRNA at temperatures up to 55 C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases TM The SuperScript II RT in this kit is provided at an optimal concentration and used at an optimal temperature for first strand cDNA synthesis T7 Oligo dT primer is a mixture of primers each consisting of a bacteriophage T7 polymerase promoter sequence followed by a string of deoxythymidylic acid dT residues followed by a nucleotide anchor that allows each primer to anneal only at the 5 end of the poly A tail of mRNA providing more efficient cDNA synthesis The sequence of the anchor varies among the primers in the mixture to allow binding to different template sequences Control HeLa RNA is included in the kit to help you deter
Download Pdf Manuals
Related Search