SuperScript Plus Indirect cDNA Labeling System
Contents
1. SuperScript III Reverse Transcriptase After a purification step to remove unincorporated nucleotides the amino modified cDNA is coupled with a monoreactive N hydroxysuccinimide NHS ester fluorescent dye included in the kit either Alexa Fluor 555 succinimidyl ester or Alexa Fluor 647 succinimidyl ester A final purification step removes any unreacted dye and the fluorescently labeled cDNA is ready for hybridization to microarrays This system uses 5 20 ug of total RNA or 0 4 2 ug of mRNA as starting material Catalog nos L1014 05 and L1014 06 include a Purification Module containing Low Elution Volume Spin Cartridges that yield a highly pure highly concentrated sample Optimized reagents and protocol ensure highly robust and reproducible labeling reactions TM e SuperScript III Reverse Transcriptase in the first strand synthesis reaction ensures high specificity and high yields of cDNA as well as more full length cONA e Use of two amino modified nucleotides in the cDNA synthesis reaction results in a greater incorporation of fluorescent dye an even distribution of fluorescent signal and higher signal intensity with small amounts of starting material e Alexa Fluor dyes provide higher correlation coefficients signal intensities and signal to background ratios than other labeling dyes e System includes all major reagents and materials for preparing Alexa Fluor labeled cDNA TM SuperScript III Reverse Tr2. by Acid Guanidinium Thiocyanate Phenol Chloroform Extraction Anal Biochem 162 156 159 De Risi J Penland L Brown P O Bittner M L Meltzer P S Ray M Chen Y Su Y A Trent J M 1996 Use of a cDNA microarray to analyse gene expression patterns in human cancer Nature Genet 14 457 460 Eisen M B Brown P O 1999 DNA arrays for analysis of gene expression Methods Enzymol 303 179 205 Gerard G F 1994 Inhibition of SuperScript II Reverse Transcriptase by Common Laboratory Chemicals Focus 16 102 103 Sambrook Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press 2004 2006 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 19 Notes Notes 6 invitrogen Corporate Headguarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
3. is recommended if you are using catalog no L1014 04 Ordering information is provided on page vii The following items are supplied by the user e Microcentrifuge e Vortex mixer The following items are supplied in the Purification Module e DEPC treated water e Low Flution Volume Spin Cartridges preinserted into collection tubes e Amber collection tubes e Binding Buffer prepared with isopropanol as described on page vi e Wash Buffer prepared with ethanol as described on page vi Continued on next page Purifying the First Strand cDNA continued Purification Procedure Use the following procedure to purify the first strand cDNA using the components of the Purification Module Cat nos L1014 05 and L1014 06 1 Add 700 pl of Binding Buffer prepared with isopropanol as described on page vi to the reaction tube containing the first strand cDNA from Hydrolysis and Neutralization Step 3 page 7 Vortex briefly to mix Each Low Elution Volume Spin Cartridge is preinserted into a collection tube For multiple reactions clearly label each collection tube and then load the cDNA Binding Buffer solution directly onto the Spin Cartridge Centrifuge at 3 300 x g in a microcentrifuge for 1 minute Remove the collection tube and discard the flow through Place the Spin Cartridge in the same collection tube and add 600 ul of Wash Buffer prepared with ethanol as described on page vi to the column Centrifuge at maximum speed f
4. 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 RNase 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 treated rinse the tubes with sterile distilled water and autoclave the tubes You can use RNase AWAY Reagent a non toxic solution available from Invitrogen see page vii to remove RNase contamination from surfaces For further information on controlling RNase contamination see Ausubel et al 1994 and Sambrook et al 1989 Isolating RNA This system is optimized for use with 5 20 pg total RNA or 0 4 2 ug of mRNA Lower amounts of starting material may be used but may result in lower hybridization signals 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 vii After you have isolated the RNA check the quality of your RNA preparation as described on the following page Co
5. 1 and K3100 02 has been tested with this kit and is recommended if you are using catalog no L1014 04 Ordering information is provided on page vii The following items are supplied by the user Microcentrifuge e Vortex mixer The following items are supplied in the Purification Module DEPC treated water Low Elution Volume Spin Cartridges pre inserted into collection tubes Amber collection tubes Binding Buffer prepared with isopropanol as described on page vi Wash Buffer prepared with ethanol as described on page vi Use the following procedure to purify the labeled cDNA using the components of the Purification Module Cat nos L1014 05 and L1014 06 1 Add 700 ul of Binding Buffer prepared with isopropanol as described on page vi to the reaction tube containing the labeled cDNA from Coupling Procedure Step 5 page 10 Vortex briefly to mix 2 Each Low Elution Volume Spin Cartridge is preinserted into a collection tube For multiple reactions clearly label each collection tube and then load the cDNA Binding Buffer solution directly onto the Spin Cartridge 3 Centrifuge at 3 300 x g in a microcentrifuge for 1 minute Remove the collection tube and discard the flow through 4 Place the Spin Cartridge in the same collection tube and add 600 pl of Wash Buffer prepared with ethanol as described on page vi to the column 5 Centrifuge at maximum speed for 30 seconds Remove the collection tube and discard th
6. 1014 04 30 Core and Dye only L1014 05 10 Core Dye and Purification L1014 06 30 Core Dye and Purification Shipping and The Core Module and Dye Module are shipped on dry ice and the Purification Storage Module is shipped at room temperature Upon receipt store the components of the Core and Dye Modules at 20 C and store the components of the Purification Module at room temperature Core Module Store at 20 C TTT TT SuperScript III Reverse 400 U pl in 20 pl 60 pl Transcriptase 20 mM Tris HCl pH 7 5 100 mM NaCl 0 1 mM EDTA 1 mM DTT 0 01 v v NP 40 50 v v glycerol 5X First Strand Buffer 250 mM Tris HCl pH 8 3 room 60 ul 200 ul temp 375 mM KCl 15 mM MgCl Dithiothreitol DTT 0 1 M DTT in water 250 ul 250 ul dATP dGTP dCTP dTTP one 15 pl 45 pl aminoallyl modified nucleotide and one aminohexyl modified nucleotide in Fe treated water 2X 2X Coupling Buffer 2X Coupling Buffer 50 pl 300 ul Anchored Oligo dT 2 2 5 ug s in DEPC treated water 20 pl 60 pl primer ao Ja av BET ernennen Warr 2mw ECC Continued on next page Vv Kit Contents and Storage continued Dye Module Store at 20 C Alexa Fluor 555 60 ug dried down dye per vial 5 vials 3x5 vials Reactive Dye Pack Alexa Fluor 647 60 ug dried down dye per vial 5vials 3x5 vials Reactive Dye Pack Purification Module Store at room temperature This module is included with Catalog Numbers L1014 05 and
7. Additional international offices are 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 18 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 expiratio
8. Invitrogen SuperScript Plus Indirect CDNA Labeling System For generating fluorescently labeled cDNA using Alexa Fluor dyes for use in microarray screening Catalog nos L1014 04 L1014 05 and L1014 06 Version D 04 January 2011 25 0794 ii Table of Contents Kit Contents and Storage i s um sn A A e de dre e e a k menee en v Accessory PTO US is Sa is e ER nn A A ea NS vii A O SERRA AE 1 MethodS sin saunaan paon m aa epa cel ud ta A tae Ara UA ama neato Se t a eh Teena neta 4 Isolattng RNA nales S 4 First Strand cDNA Synthesis it o ea e e E aaae aa aeee EAE Ee aaa EEEE 6 Purifying the First Strand cDNA nsr iee sea i e E AE E oe EEEE a T E a TEn 8 Coupling with Fluorescent Pye moi oo San enter a A 10 Purifying the Fluorescently Labeled cDNA cmcmeeusmsmsmssas tutut matemaattisten 11 Hybridization ns eh mot me mas Hilfe ANK etuna ah 13 A nissa vk i vihana Er ea sane ete EAA ravi matat aaa ma aa maa KE ra 14 Assessing Labeling Efficiency sainia eia o a ieee Eea Ee ea EEEo Lo EEREN EERE nara eee nene 14 Troubleshooting OS SS 15 No nenn Heu R KOHA SESSA Ea 17 Technical Support sia sto iaa 18 References nn TR LEE 19 iii iv Kit Contents and Storage Kit Sizes and All versions of the SuperScript Plus Indirect cDNA Labeling System are Modules supplied with a Core Module and a Dye Module Catalog nos L1014 05 and L1014 06 also include a Purification Module Cat no Number of Labeling Reactions Modules L
9. L1014 06 eine Low Elution Volume Spin Cartridges with 2x11 columns 6 x 11 columns collection tubes Binding Buffer must be combined with 100 2 x 5 5 ml 2x 18 ml isopropanol to create final buffer see below Wash Buffer must be combined with 100 ethanol 2x 2ml 2x 5 ml to create final buffer see below Amber collection tubes 2 x 11 tubes 6 x 11 tubes Preparing Binding Buffer with Isopropanol Preparing Wash Buffer with Ethanol vi The Binding Buffer supplied with the Purification Module must be mixed with 100 isopropanol prior to use Add the amount of isopropanol indicated below directly to the bottle of Binding Buffer to create the final buffer Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the isopropanol 10 rxn kit 30 rxn kit Binding Buffer 5 5 ml entire bottle 18 0 ml entire bottle 100 Isopropanol 2 0 ml 6 5 ml Final Volume 7 5 ml 24 5 ml Store the Binding Buffer prepared with isopropanol at room temperature The Wash Buffer supplied with the Purification Module must be mixed with 100 ethanol prior to use Add the amount of ethanol indicated below directly to the bottle of Wash Buffer to create the final buffer Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the ethanol 10 rxn kit 30 rxn kit Wash Buffer 2 ml entire bottle 5 ml entire bottle 100 Ethanol 8ml 20 ml Final Volume 10 ml 25 ml Store the Wash Buffer
10. actic 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 the above patents 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 Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0354 CyDye and Cy are trademarks of Amersham Biosciences 17 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 FAOs 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
11. alization reaction is incorrect This affects the pH of the reaction and therefore may affect binding to the column cDNA has been lost during Measure the amount of cDNA in the control purification reaction before and after purification Follow the purification procedure without modifications Continued on next page 15 Troubleshooting continued Amount of coupled Reaction tubes have been Avoid direct exposure of the labeling reaction to dye in the control exposed to light light Use amber tube provided in the kit for reaction is low collection of the final product lt 24 pmoles and or fluorescence of labeled cDNA is low Dye solution has been exposed Repeat the labeling reaction with a fresh mixture to light of dye being careful to avoid direct exposure to light DMSO used to prepare dye Prepare a new mixture of dye using fresh DMSO mixture was contaminated Carefully follow the instructions for storing and with water handling DMSO in the Caution on page 10 Inefficient labeling due to Follow all purification steps carefully and without improper purification modification 2X Coupling Buffer was not Store 2X Coupling Buffer at 20 C stored properly 16 Purchaser Notification Limited Use Label License No 149 Indirect cDNA Labeling Notice Limited Use Label License No 223 Labeling and Detection Technology Trademarks Unless indicated otherwise purchase of this product may not convey to
12. anscriptase 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 III RT in this kit is provided at an optimal concentration and used at an optimal temperature for incorporating amino modified nucleotides in first strand cDNA synthesis Continued on next page Overview continued Experimental The flow chart below outlines the experimental steps of the system Outline Isolate total RNA v Perform first strand cDNA synthesis using SuperScript III RT and amino modified dNTPs Purify amino modified cDNA using Purification Purify amino modified cDNA using method of Module Cat Nos L1014 05 and L1014 06 choice Cat No L1014 04 Perform the fluorescent dye coupling reaction y y Purify the labeled cDNA using Purification Purify the labeled cDNA using method of choice Module Cat Nos L1014 05 and L1014 06 Cat No L1014 04 up Ready to hybridize Alexa Fluor 555 The Alexa Fluor 555 and Alexa Fluor 647 dyes included in this kit are and Alexa Fluor compatible with commonly used microarray scanners and provide greater 647 Reactive Dyes signal correlation R values than the spectrally similar Cy 3 and Cy 5 dye pair
13. e 1 5 ml RNase free microcentrifuge tubes The following materials are supplied in the kit e Anchored Oligo dT 2 primer e Random hexamers for mRNA starting material only e dNTP mix including amino modified nucleotides e 5X First Strand buffer e 0 1MDTT e RNaseOUT e SuperScript III RT e DEPC treated water e 10 ug of Control HeLa RNA per reaction optional see page 3 RNaseOUT Recombinant RNase Inhibitor has been included in the system to Note safeguard against degradation of target RNA due to ribonuclease contamination of the RNA preparation Continued on next page First Strand cDNA Synthesis continued First Strand cDNA The following procedure is designed to convert 5 20 ug of total RNA or 0 4 2 ug of mRNA into first strand cDNA Synthesis Reaction Hydrolysis and Neutralization Note If you are setting up a control reaction recommended for first time users use 10 ul of the Control HeLa RNA supplied in the kit 1 ug yl 1 2 Mix and briefly centrifuge each component before use Prepare each reaction as follows in a 1 5 ml RNase free tube Component Volume 5 20 ug total RNA or 0 4 2 ug mRNA X pl Anchored Oligo dT Primer 2 5 pg pl 2 ul Random hexamers only if using mRNA 1 pl DEPC treated water to 18 ul For mRNA use both anchored oligo dT and random hexamers For total RNA use only 2 pl of anchored oligo dT 2 Incubate tubes at 70 C for 5 minutes and then place on ice for a
14. e 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 Follow the steps below to couple Alexa Fluor dye to the amino modified first strand cDNA Use only the DMSO provided with this kit 1 Dry the purified first strand cDNA from Step 9 page 9 in a speed vac at medium heat until the volume is reduced to 3 pl 2 Add 5 pl of 2X Coupling Buffer to the tube 3 Add 2 pl of DMSO directly to a vial of Alexa Fluor Reactive Dye to resuspend the dye Vortex thoroughly and then spin briefly to collect the contents 4 Add the DMSO dye solution to the tube from Step 2 and vortex to mix thoroughly 5 Incubate the tube at room temperature in the dark for 1 2 hours Reaction can be stored overnight if necessary Proceed to Purifying the Fluorescently Labeled cDNA on the next page Purifying the Fluorescently Labeled cDNA Introduction Note Before Starting Purification Procedure Catalog nos L1014 05 and L1014 06 include a Purification Module developed for use with the system Follow the procedure below to purify the labeled cDNA using this module Catalog no L1014 04 does not include a Purification Module Use your preferred purification method instead of the following procedure and then continue to hybridization The PureLink PCR Purification System K3100 0
15. e flow through 6 Placethe Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer Remove the collection tube and discard Procedure continued on next page Continued on next page 11 Purifying the Fluorescently Labeled cDNA continued Purification Procedure continued Note on cDNA Purity 12 Procedure continued from previous page 7 Place the Spin Cartridge onto a new amber collection tube supplied in the kit 8 Add 20 pl of DEPC treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute 9 Centrifuge at maximum speed for 1 minute to collect the purified cDNA The eluate contains your purified labeled cDNA The sample may be stored at 20 C for up to one week prior to hybridization Avoid freeze thawing To determine the efficiency of the labeling reaction proceed to Assessing Labeling Efficiency page 14 Because of the high purity of the cDNA from the Low Elution Volume Spin Cartridges included with catalog nos L1014 05 and L1014 06 the yield and picomole dye incorporation calculations will be more accurate than with other purification methods In the 1 2 E Gel below Lanes 1 and 2 contain Alexa Fluor 555 labeled cDNA purified using the Low Elution Volume Spin Cartridges and Lanes 3 and 4 contain Alexa Fluor 555 labeled cDNA purified using columns from another manufacturer The labeled cDNA app
16. ears as smear from 500 5 000 bp The large band at the bottom of Lanes 3 and 4 is unincorporated dye that was not removed by the other manufacturer s purification column Such material would be included in the picomole dye incorporation calculations resulting in an incorporation level that is higher than theoretically possible For this reason we strongly recommend using the purification columns provided with catalog nos L1014 05 and L1014 06 1kbladder 1 2 3 4 5000 Hybridization Hybridization After purification you are ready to use the labeled cDNA in any application of choice including glass microarray hybridization Follow the preparation and hybridization instructions for your specific application 13 Appendix Assessing Labeling Efficiency Introduction You can use UV visible spectroscopy scanning to measure the amount of labeled cDNA and dye incorporation The expected amounts using the Control HeLa RNA provided in the kit are shown below Calculating the To calculate the amount of labeled cDNA using a UV visible spectrophotometer Results 1 Transfer a volume of purified labeled cDNA from step 9 page 12 to a clean cuvette Use an appropriate volume for your spectrophotometer Add DEPC treated water to the cDNA if you need to increase the volume of the eluate for your spectrophotometer Note The labeled DNA must be purified as described on page 11 before scanning as any unincorporated dye will interf
17. ere with the detection of labeled DNA Blank the spectrophotometer using DEPC treated water and then scan the sample at 240 800 nm Wash each cuvette thoroughly between samples Calculate the yield of cDNA using the following formula cDNA ng A260 A320 x 37 ng pl x volume in pl Calculate the amount of fluorescent dye using the following formulas Alexa Fluor 555 pmole As55 Ag50 0 15 x volume in ul Alexa Fluor 647 pmole A650 A750 0 24 x volume in pl Calculate the base to dye ratio using the following formulas Base dye ratio for Alexa Fluor 555 A 260 A 320 Asss A650 x 0 04 x 150 000 Asss Asso x 8 919 Base dye ratio for Alexa Fluor 647 A 260 A 320 Asso A750 x 0 x 239 000 Acso A750 x 8 919 The number of dye molecules per 100 bases is calculated using the formula 100 base dye ratio Expected If you prepare a control reaction using 10 ug of Control HeLa RNA as starting Amounts Using material the following amounts are expected Control DNA Labeled DNA Incorporated Dye Dyes Molecules 100 Bases gt 250 ng gt 24 pmole 22 50 If you do not obtain these amounts see Troubleshooting on page 15 14 Troubleshooting 285 and 185 bands Too little RNA loaded on the Be sure to load at least 250 ng of RNA for are not observed gel analysis arter isolation RNA is degraded due to Follow the guidelines on page 4 to avoid RNase total RNA and RNase activit
18. improving the resolution of two color microarray gene expression assays The exceptionally bright Alexa Fluor dyes are also insensitive to pH and are highly water soluble The table below shows the excitation and emission maxima and color of each dye Dye Excitation Emission nm Color Alexa Fluor 555 555 565 Orange Fluorescent Alexa Fluor 647 650 670 Far Red Fluorescent Anchored Anchored oligo dT 2o primer is a mixture of 12 primers each consisting of a Oligo dT zo string of 20 deoxythymidylic acid dT residues followed by two additional nucleotides represented by VN where V is dA dC or dG and N is dA dC dG or dT The VN anchor allows the primer to anneal only at the 5 end of the poly A tail of mRNA providing more efficient CDNA synthesis for labeling applications Continued on next page Overview continued Materials Supplied by the User Control Reaction Product Qualification In addition to the kit components you should have the following items on hand before using the SuperScript Indirect cDNA Labeling System e Vortex mixer e Microcentrifuge e Aerosol resistant pipette tips e Water baths or incubator e 1NNaOH e 1NHCI e Sterile microcentrifuge tubes e 100 Isopropanol e 100 Ethanol e 75 Ethanol We recommend performing the labeling procedure using the Control HeLa RNA included in the system to determine the efficiency of the labeling reaction The section on First Strand cDNA S
19. n date No warranty is applicable unless all 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 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 Chirgwin J M Przybyla A E MacDonald R J and Rutter W Z 1979 Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonucleases Biochem 18 5294 5299 Chomczynski P and Sacchi N 1987 Single Step Method of RNA Isolation
20. ntinued on next page Isolating RNA continued Checking the RNA Quality Storing RNA To check RNA quality analyze 500 ng of RNA by agarose ethidium bromide gel electrophoresis You can use a regular 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 285 and 185 rRNA will appear as bands at 4 5 kb and 1 9 kb respectively The 285 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 285 band with an accumulation of low molecular weight RNA on the gel are indications that the RNA may be degraded which will decrease the labeling efficiency If you do not detect any RNA you will need to repeat RNA isolation Refer to the Troubleshooting section on page 15 After preparing the RNA we recommend that you proceed directly to First Strand cDNA Synthesis on page 6 Otherwise store the RNA at 80 C First Strand cDNA Synthesis Introduction After you have isolated RNA and checked the guality of your RNA preparation you are ready to synthesize cDNA Before Starting The following materials are supplied by the user e 5 20 pg total RNA or 0 4 2 ug mRNA e 1NNaOH e 1NHCI e Water baths heating block or incubator set at 46 C and 70 C e Ice
21. or 30 seconds Remove the collection tube and discard the flow through Place the Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer Remove the collection tube and discard Place the Spin Cartridge onto a new amber collection tube supplied in the kit Add 20 pl of DEPC treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute Centrifuge at maximum speed for 1 minute to collect the purified first strand cDNA The eluate contains your purified cDNA Proceed directly to Coupling with Fluorescent Dye on the next page Coupling with Fluorescent Dye Introduction Before Starting Important Coupling Procedure 10 After cDNA synthesis and purification you are ready to couple the amino modified cDNA with Alexa Fluor dye The following items are supplied by the user e Microcentrifuge e Vortex mixer The following items are provided in the kit e DMSO e 2X Coupling Buffer e Alexa Fluor 555 Reactive Dye Pack 60 ug per vial Alexa Fluor 647 Reactive Dye Pack 60 ug per vial 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 dy
22. prepared with ethanol at room temperature Accessory Products Additional Many of the reagents in the SuperScript Indirect cDNA Labeling System as Products well as additional reagents that may be used with this system are available separately from Invitrogen Ordering information is provided below SuperScript Indirect cDNA Labeling System 10 reactions L1014 01 30 reactions L1014 02 30 reactions w o L1014 03 purification module Purification System TRIzol Reagent 100 ml 15596 026 m issa FastTrack 2 0 mRNA Isolation Kit 6 reactions K1593 02 TTT radin kasan RNaseOUT Recombinant Ribonuclease 5000 units 10777 019 Inhibitor PureLink PCR Purification System 50 reactions K3100 01 250 reactions K3100 02 Alexa Fluor 555 reactive dye decapack A32756 Alexa Fluor 647 reactive dye decapack A32757 Alexa Fluor 555 and Alexa Fluor 647 reactive dye decapacks Yeast tRNA 25 mg 15401 011 ps Sm iina UltraPure 20X SSC 15557 044 UltraPure 20x SSPE 15591 043 vii viii Overview Introduction Advantages of the System Advantages of SuperScript III Reverse Transcriptase The SuperScript Plus Indirect cDNA Labeling System is a highly efficient system for generating fluorescently labeled cDNA for use on microarrays in gene expression studies It uses an aminoallyl modified nucleotide and an aminohexyl modified nucleotide together with other dNTPs in a cDNA synthesis reaction with
23. t least 1 minute Add the following to each tube on ice Component Volume 5X First Strand buffer 6 ul 0 1 M DTT 1 5 pl dNTP mix including amino modified nucleotides 1 5 ul RNaseOUT 40 U ul 1 ul SuperScript III RT 400 U yl _2ul Final Volume 30 ul Mix gently and collect the contents of each tube by brief centrifugation Incubate tube at 46 C for 2 3 hours Note A 3 hour incubation results in 20 30 higher cDNA yield than a 2 hour incubation After incubation proceed directly to Alkaline Hydrolysis and Neutralization below After cDNA synthesis immediately perform the following hydrolysis reaction to degrade the original RNA 1 2 3 Add 15 ul of 1 N NaOH to each reaction tube from Step 5 above Mix thoroughly Incubate tube at 70 C for 10 minutes Add 15 ul of 1 N HCl to neutralize the pH and mix gently Proceed to Purifying the First Strand cDNA on the next page Purifying the First Strand cDNA Introduction Note Before Starting Catalog nos L1014 05 and L1014 06 include a Purification Module developed for use with the system Follow the procedure in this section to purify the amino modified first strand cDNA using this module Catalog no L1014 04 does not include a Purification Module Use your preferred purification method and then continue to Coupling with Fluorescent Dye on page 10 The PureLink PCR Purification System K3100 01 and K3100 02 has been tested with this kit and
24. the purchaser a license to practice any claim in any other patent including but not limited to United States and foreign patents Users of this product should determine if any license is required under these or any other patents 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 to not 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 its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophyl
25. y contamination agarose gel electrophoresis Yield of cDNA is Temperature too high during Perform the cDNA synthesis at 46 C low cDNA synthesis Incorrect reaction conditions Verify that all reaction components are included used in the reaction and use reagents provided in the system Use a fresh sample for RNA isolation Verify the reaction conditions using the control RNA provided in the kit Concentration of template Increase the concentration of template RNA Use RNA is too low at least 5 ug of total RNA or 0 4 ug of mRNA Poor quality RNA used or Check the quality of your RNA preparation see RNA is degraded page 5 If RNA is degraded use fresh RNA RNase contamination Use the RNaseOUT included in the kit to prevent RNA degradation RTinhibitors are present in Inhibitors of RT include SDS EDTA guanidinium your RNA sample chloride formamide sodium phosphate and spermidine Gerard 1994 Remove inhibitors from your RNA sample by performing an additional 70 ethanol wash after ethanol precipitation during RNA isolation and purification Test for the presence of inhibitors by mixing 1 ug of control RNA with 25 ug total RNA or 1 ug mRNA and compare the yields of first strand synthesis Improper storage of Store the enzyme at 20 C SuperScript III RT Concentration of NaOH Verify the concentration of NaOH and HCL and and or HCl used in the repeat the reaction if necessary hydrolysis and neutr
26. ynthesis page 6 describes how to set up the control reaction and page 14 has equations for calculating the efficiency of the labeling procedure This kit was verified in replicate labeling reactions using 10 pg of Control HeLa RNA 2 ul of 2 5 pg ul anchored oligo dT 2 primer and amino modified dNTP mix for cDNA synthesis For the coupling step Alexa Fluor 555 or Alexa Fluor 647 dyes were used After purification the labeled cDNA was scanned to read the full absorbance spectrum from 240 800 nm The amount of coupled dye was calculated using the formulas on page 14 In addition each reaction was run ona 1 2 E Gel to determine the quality of the product 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 mRNA using a method of choice The quality of the RNA is critical for successful labeling and hybridization The Important presence of contaminants in the RNA may significantly increase background fluorescence in your microarrays Carefully follow the recommendations below to prevent RNase 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
Download Pdf Manuals
Related Search