NCode miRNA Amplification System
Contents
1. 24 Isolating Small Amounts of Small RNA 25 Purchaser Notification 27 Technical Service 28 References 29 iv v Kit Contents and Storage Shipping and Storage The NCode miRNA Amplification System is shipped in two modules The miRNA Amplification Module is shipped on dry ice while the Purification Module is shipped at room temperature Store the components of the miRNA Amplification Module at 20 C and the components of the Purification Module at room temperature miRNA Amplification Module Components should be stored at 20 C Reagents are provided for 20 amplification reactions Component Amount 10X miRNA Reaction Buffer 110 l 25 mM MnCl2 100 l Poly A Polymerase 20 l Oligo dT 24V 15 l 10 mM ATP 20 l 5X First Strand Buffer 250 mM Tris HCl pH 8 3 375 mM KCl 15 mM MgCl2 200 l 0 1 M DTT 100 l2. 10 Reverse Transcription of Tailed miRNA 12 Purification and Concentration of First Strand cDNA 14 Tailing of First Strand cDNA 16 T7 Promoter Synthesis 17 In Vitro Transcription 18 Purifying senseRNA 20 Quantifying senseRNA 22 Labeling and Hybridization 23 Appendix 24 Troubleshooting
3. Microcentrifuge Purification Procedure Use the following procedure to purify and concentrate the cDNA 1 Insert a cDNA Ultrafiltration Column into a cDNA Ultrafiltration Tube You will need a separate column and tube for each sample processed 2 Pipette the 100 l of cDNA from Step 11 page 13 onto the membrane in the center of the column Do not touch the membrane with the pipette tip 3 Secure the tube cap and insert the assembly in a centrifuge Note Align the cap strap toward the center of the rotor and be sure to counterbalance the rotor with a similar device 4 Centrifuge for 6 minutes at 13 000 g 5 Add 200 l of 1X TE buffer to the column without touching the membrane Pipette the buffer up and down 5 times 6 Secure the tube cap as before and centrifuge for 6 minutes at 13 000 g 7 Carefully separate the column from the tube and discard the flow through Insert the column in the same tube Procedure continued on next page Continued on next page Assembled column tube Unassembled column tube 15 Purification and Concentration of First Strand cDNA continued Purification Procedure continued Procedure continued from previous page 8 Add 200 l of 1X TE buffer to the column without touching the membrane Gently pipette the buffer up and down 5 times 9 Secure the tube cap as before and centrifuge for 6 minutes at 13 000 g 10 Carefully separate the column
4. 10 mM dNTP Mix 100 l RNaseOUT Recombinant Ribonuclease Inhibitor 40 U l 25 l SuperScript III Reverse Transcriptase 200 U l 40 l DEPC treated Water 2 ml 10 mM dTTP 80 l Terminal Deoxynucleotidyl Transferase 40 l Klenow 20 l T7 Template Oligo 40 l 100 mM ATP 30 l 100 mM CTP 30 l 100 mM GTP 30 l 100 mM UTP 30 l 10X T7 Reaction Buffer 80 l T7 Enzyme Mix includes T7 RNA Polymerase in a proprietary formulation 140 l cDNA Purification Module Components should be stored at room temperature Columns and tubes are provided for 20 purifications Item Amount cDNA Ultrafiltration Columns 20 columns cDNA Ultrafiltration Tubes 2 20 tubes vi Accessory Products Additional Products The NCode miRNA Amplification System is part of an integrated microRNA expression profiling system that includes miRNA isolation amplification purification quantification labeling and array hybridization components Additional products are available separately from Invitrogen Ordering information is provided below For more information visit our Web site at www invitrogen com or contact Technical Service page 28 Product Quantity Catalog no PureLink miRNA Isolation Kit 25 preps K1570 01 PureLink Micro to Midi Total RNA Purification System 50 isolations 12183 018 Quant iT Ribogreen RNA Assay Kit 200 2 000 cuvette assays R 11490
5. 11 Polyadenylation of miRNA continued Polyadenylation Procedure Use the following procedure to add poly A tails to up to 30 ng of enriched miRNA 1 Following quantification of the enriched miRNA as described on page 9 prepare an 18 l volume of sample containing 30 ng of enriched miRNA using one of the following methods Aliquot up to 18 l of eluate containing 30 ng of enriched miRNA into an RNase free microcentrifuge tube If necessary add DEPC treated water to increase the volume to 18 l OR If the sample is extremely dilute concentrate an amount of eluate containing 30 ng of enriched miRNA in a SpeedVac Concentrator at low heat to a final volume of 18 l Note The elution volume from the PureLink miRNA Isolation Kit is 50 100 l Transfer to an RNase free microcentrifuge tube 2 Dilute the 10 mM ATP see Important note on the previous page as follows For enriched miRNA samples between 1 and 30 ng dilute a volume of 10 mM ATP in 1 mM Tris pH 8 0 according to the following formula ATP dilution factor 5000 ____ ng of enriched miRNA Example If you are starting with 5 ng of miRNA the ATP dilution factor is 5000 5 ng 1000 Dilute the 10 mM ATP 1 1000 by adding 1 l of 10 mM ATP to 999 l of 1 mM Tris pH 8 0 For enriched miRNA samples less than 1 ng dilute the 10 mM ATP 1 5000 in 1 mM Tris pH 8 0 3 Add the following at room temperature to the tube
6. miRNA Labeling System The NCode miRNA Labeling System is a robust and efficient system for labeling and hybridizing miRNA to NCode microarrays for expression profiling analysis Using this kit you ligate a short highly specific tag sequence to each miRNA and then hybridize highly fluorescent Alexa Fluor dye molecules to the tagged miRNA The high specificity of the binding sequence and high fluorescence of the dye molecules ensure maximum signal and strong signal correlations The NCode Multi Species miRNA Microarray V2 consists of 5 Corning Epoxide Coated Glass Slides each printed with optimized probe sequences targeting all of the known mature miRNAs in miRBase Release 9 0 http microrna sanger ac uk for human mouse rat D melanogaster C elegans and Zebrafish The probes were designed using an algorithm that generates miRNA sequences with enhanced hybridization properties Goff et al 2005 Each slide comes blocked and ready to use The NCode Multi Species miRNA Microarray Probe Set includes the probe sequences provided on the microarray listed above dried down in 384 well plates at 500 pmoles per well and ready for printing on standard DNA microarray surfaces The NCode Multi Species miRNA Microarray Control is a synthetic 22 nucleotide miRNA sequence that has been designed and screened as a positive control for use with NCode system This sequence has been tested for cross reactiv
7. If you are starting with total RNA analyze it by agarose ethidium bromide gel electrophoresis prior to isolation of small RNA 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 Condensation formed in the in vitro transcription reaction tube If condensation forms inside the tube during incubation spin the tube briefly to remix the components and perform the reaction in a different incubator Note that the incubator must heat the tube evenly to avoid condensation on the tube lid Do not use heat blocks or water baths Poor quality RNA used or RNA is degraded Check the quality of your RNA preparation see page If RNA is degraded use fresh RNA RNase contamination Use the RNaseOUT included in the kit to prevent RNA degradation RT enzyme inhibitors are present in your RNA sample Inhibitors of RT enzymes include SDS EDTA guanidinium chloride formamide sodium phosphate and spermidine Gerard 1994 Test for the presence of inhibitors by mixing 1 g of Control HeLa RNA with 25 g total RNA or 1 g mRNA and compare the yields of senseRNA amplification Reagents were not properly mixed before first strand synthesis Repeat the procedure being careful to briefly vortex and centrifuge each reagent before first strand
8. PCR may be performed on specific miRNA sequences to determine the level and specificity of the amplification reaction prior to array hybridization The NCode SYBR Green miRNA qRT PCR Kit provides qualified reagents for the sensitive detection and quantification of miRNA sequences 23 Labeling and Hybridization Introduction After you have purified the senseRNA and determined the yield you are ready to label the sample using the NCode miRNA Labeling System For a description of this and other NCode products see page 3 Note that you do not need to add a poly A tail to the amplified senseRNA prior to labeling with the NCode miRNA Labeling System The senseRNA is already tailed Proceed directly to the Ligation of the Capture Sequence protocol in the NCode miRNA Labeling System manual Amount of senseRNA We recommend using 1 5 g of senseRNA in each labeling reaction 24 Appendix Troubleshooting Problem Cause Solution Problems with the small RNA isolation procedure Use the PureLink miRNA Isolation Kit for optimal results See your miRNA isolation kit manual for additional troubleshooting information Note that we do not recommend using total RNA in place of small RNA with this kit Yield of enriched miRNA is low Degraded starting material Follow the guidelines on page 6 to prevent RNase contamination of the RNA preparation Always use fresh samples or samples frozen at 80 C
9. designed T7 template oligo Finally you perform an in vitro transcription reaction with an overnight incubation to generate the amplified senseRNA Advantages of the System Optimized reagents and protocol ensure highly robust and reproducible reactions SuperScript III Reverse Transcriptase in the first strand synthesis reaction ensures high specificity and yields of cDNA System generates amplified miRNA in the sense orientation for direct compatibility with microarray probe sequences System includes all major reagents and materials for preparing amplified senseRNA for subsequent labeling and detection MicroRNAs MicroRNAs miRNAs are a recently discovered class of small 19 23 nucleotide non coding RNA molecules They are cleaved from hairpin precursors and are believed play an important role in translation regulation and degradation of target mRNAs by binding to partially complementary sites in the 3 untranslated regions UTRs of the message Lim 2003 Recent experimental evidence suggests that the number of unique miRNAs in humans could exceed 800 though several groups have hypothesized that there may be up to 20 000 non coding RNAs that contribute to eukaryotic complexity Bentwich et al 2005 Imanishi et al 2004 Okazaki et al 2002 Though hundreds of miRNAs have been discovered in a variety of organisms little is known about their cellular function They have been implicated in regulation
10. from the tube and discard the tube with the flow through 11 Add 5 l of 10 mM Tris pH 8 0 to the column membrane without touching the membrane Gently tap the side of the column to mix 12 Carefully place the column upside down in a new cDNA Ultrafiltration Tube 13 Secure the tube cap and centrifuge for 3 minutes at 13 000 g The eluate collected in the tube is your purified concentrated cDNA 14 The volume of cDNA in the tube should be 5 10 l If necessary bring the volume up to 10 l with DEPC treated water Proceed to Tailing of First Strand cDNA next page Column inserted upside down in tube 16 Tailing of First Strand cDNA Introduction In this step you add a poly T tail to the 3 end of the purified cDNA using Terminal Deoxynucleotidyl Transferase and dTTP Before Starting The following items are supplied in the miRNA Amplification Module Terminal Deoxynucleotidyl Transferase 10 mM dTTP 10X miRNA Reaction Buffer DEPC treated water The following items are supplied by the user 1 5 ml RNase free microcentrifuge tubes Incubator or thermal cycler at 80 C Heat block at 37 C Ice Microcentrifuge Tailing Procedure Perform the following tailing reaction for each vial of purified cDNA 1 Cap the tube containing the purified cDNA from Step 14 page 15 and heat treat at 80 C for 10 minutes Chill on ice for 1 2 minutes bri
11. 1 000 ng ml Use 1 l of purified senseRNA in the quantitation reaction See the product information sheet for each kit for detailed protocols Determining Yield Using A260 Absorbance The following general protocol may be used to calculate the yield of the senseRNA by measuring A260 absorbance 1 Aliquot 1 l of the purified senseRNA into a clean cuvette in most cases the amount of senseRNA from the purification procedure is small enough that further dilution is not necessary 2 Scan the sample at 260 nm using a UV visible spectrophotometer Be sure to blank the spectrophotometer using the sample elution buffer e g DEPC treated water before the reading 3 Note The A260 reading should fall within the standard specification for the spectrophotometer typically 0 1 1 0 OD If it falls outside this range dilute the sample and re scan If the A260 reading is too low use a lower dilution if it s too high use a higher dilution 4 Transfer the sample back into the Recovery Tube for storage 5 Calculate the yield of senseRNA using the formula below Total senseRNA yield g ml A260 40 g ml RNA dilution factor elution volume For example if you diluted 1 l of a 100 l volume of senseRNA at 1 50 and the A260 is 0 5 then 0 5 40 g ml RNA 10 1000 g ml In a 100 l volume you would have 100 g of senseRNA Determining Yield and Specificity Using qRT PCR Quantitative RT PCR qRT
12. RediPlate 96 Ribogreen RNA Quantitation Kit 96 well plate 8 12 strip wells R 32700 NCode miRNA Labeling System 20 labeling and hybridization reactions MIRLS 20 NCode Multi Species miRNA Microarray V2 5 slides MIRA2 05 NCode Multi Species miRNA Microarray Control V2 10 l MIRAC2 01 NCode Multi Species miRNA Microarray Probe Set V2 3 384 well plates 500 pmol per well MIRMPS2 01 Quant iT Ribogreen RNA Assay Kit 200 2 000 cuvette assays R 11490 NCode SYBR Green miRNA qRT PCR Kit 10 polyadenylation 20 cDNA synthesis 100 qPCR reactions MIRQ 100 NCode SYBR GreenER miRNA qRT PCR Kit 10 polyadenylation 20 cDNA synthesis 100 qPCR reactions MIRQER 100 NCode miRNA First Strand cDNA Synthesis Kit 10 polyadenylation 20 cDNA synthesis 50 polyadenylation 100 cDNA synthesis MIRC 10 MIRC 50 RediPlate 96 Ribogreen RNA Quantitation Kit 96 well plate 8 12 strip wells R 32700 UltraPure 20X SSC 1 liter 4 liters 15557 044 15557 036 1 Introduction Overview The NCode miRNA Amplification System is a highly robust and efficient system for amplifying senseRNA molecules from minute quantities of purified microRNA miRNA to generate sufficient amounts of material for downstream research The system provides consistent and accurate 1000 fold amplification of miRNA The resulting amplified miRNA is in the sense orientation for di
13. User Manual 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 speci c contact information visit our web site at www invitrogen com NCode miRNA Amplification System For generating amplified senseRNA from small starting quantities of miRNA Catalog no MIRAS 20 Version B 12 January 2007 25 0918 ii iii Table of Contents Kit Contents and Storage v Accessory Products vi Introduction 1 Methods 6 Isolating Small RNA 6 Quantifying Small RNA 9 Polyadenylation of miRNA
14. before using this kit Ordering information for Invitrogen products listed below is provided on page vi 300 500 ng of total RNA or equivalent cells tissue for smaller amounts of starting material see purification protocol starting on page 25 PureLink miRNA Isolation Kit Invitrogen or other miRNA isolation kit PureLink Micro to Midi Total RNA Purification System Invitrogen or other column based total RNA purification system Quant iT Ribogreen RNA Assay Kit Invitrogen or RediPlate 96 Ribogreen RNA Quantitation Kit Invitrogen or capillary or other small volume spectrophotometer Optional SpeedVac or other concentrator may be required to concentrate sample prior to polyadenylation Thermal cycler with a heated lid or air incubator heat block water bath may be used for some but not all procedures Microcentrifuge Vortex mixer 1 5 ml RNase free microcentrifuge tubes RNase free pipette tips 10 mM Tris pH 8 0 0 1X TE buffer 1 mM Tris HCl 0 1 mM EDTA pH 8 0 1X TE buffer 10 mM Tris HCl 0 1 mM EDTA pH 8 0 0 5 M NaOH 50 mM EDTA 1 M Tris pH 8 0 100 ethanol 96 100 ethanol Ice Product Qualification This kit was verified using enriched miRNA in a standard amplification reaction as described in this manual Equivalent quantities of amplified miRNA and nonamplified miRNA sample were assaye
15. 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 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 prophyla
16. cDNA synthesis Yield of senseRNA is low Precipitates formed in 10X T7 Reaction Buffer Vortex the buffer after warming to room temperature to avoid precipitation If necessary briefly heat to 37 C to dissolve precipitates 25 Isolating Small Amounts of Small RNA Introduction The standard range of starting material for this kit is 300 500 ng of total RNA or equivalent cells or tissue If you are starting with smaller amounts of sample down to 50 ng of total RNA you can use the procedure in this section to isolate small RNA prior to amplification For the following procedure you can use either the cDNA Ultrafiltration Columns and Tubes provided in this kit or order columns and tubes separately from Millipore Corporation Note that this kit includes only enough cDNA Ultrafiltration Columns and Tubes to perform 20 first strand cDNA purifications described starting on page 14 If you use these columns and tubes in the following procedure you will have fewer columns and tubes with which to perform the full amplification procedure Before Starting Select one of the following cDNA Ultrafiltration Columns and Tubes from the cDNA Purification Module or Microcon YM 100 Centrifugal Filter Unit 100 columns and tubes Millipore catalog no 42413 The following additional items are supplied by the user 50 500 ng of total RNA in a volume of 70 l DEPC treated water Microcentrifuge If
17. ctic 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 28 Technical Service Web Resources Visit the Invitrogen Web site at www invitrogen com for Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc Complete technical service contact information Access to the Invitrogen Online Catalog Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 USA Tel 1 760 603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 602 6500 E mail tech_service invitrogen com Japanese Headquarters Invitrogen Japan LOOP X B
18. d by qRT PCR using primers for specific miRNA sequences Cycle thresholds CTs and fold amplification were calculated and compared to determine sequence specific amplification 6 Methods Isolating Small RNA Introduction In this step you isolate small cellular RNA molecules from biological samples General Handling of RNA When working with RNA Use disposable individually wrapped sterile plasticware Use aerosol resistant pipette tips for all procedures Use only sterile new pipette tips and microcentrifuge tubes Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin Use proper microbiological aseptic technique when working with RNA Dedicate a separate set of pipettes buffers and enzymes for RNA work Use RNase free microcentrifuge tubes 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 You can use RNase Away Reagent a non toxic solution available from Invitrogen to remove RNase contamination from surfaces For further information on controlling RNase contamination see Ausubel et al 1994 Sambrook et al 1989 Amount of Starting Material The PureLink miRNA Isolation Kit can be used to isolate small RNA molecules from 300 500 ng of total RNA or e
19. day The 4 16 hour incubation at 37 C requires the use of an air incubator Do not use a heat block water bath or thermocycler for the incubation The reaction tube must be heated evenly throughout the incubation to avoid condensation on the tube lid Heating methods that result in condensation may compromise the reaction The following reaction uses the 100 mM ATP included in the kit not the 10 mM ATP used in the polyadenylation reaction page 10 Be careful to select the vial of 100 mM ATP for use in this reaction Continued on next page 19 In Vitro Transcription continued In Vitro Transcription Procedure The following procedure is for a single reaction For multiple reactions prepare a master mix with a 5 10 overage to enable accurate pipetting 1 Thaw the individual 100 mM NTPs see Important note on the previous page and T7 Enzyme Mix at room temperature and hold at room temperature until use 2 Thaw and warm the 10X T7 Reaction Buffer at 37 C in an air incubator then vortex briefly to dissolve any precipitates Hold at room temperature 3 Incubate the 25 l of cDNA from Step 5 page 17 at 37 C for 10 minutes to re anneal the strands 4 For each reaction add the following components to the tube of cDNA at room temperature for a final volume of 42 l Component Volume 100 mM ATP 1 5 l 100 mM CTP 1 5 l 100 mM GTP 1 5 l 100 mM UTP 1 5 l 10X T7 Reaction Buffer 4
20. e used to measure A260 absorbance After quantifying the small RNA we recommend that you proceed directly to Polyadenylation of miRNA on page 10 The RNA may be stored at 80 C if necessary 10 Polyadenylation of miRNA Introduction After you have quantified the enriched miRNA you are ready to add a poly A tail to the miRNA Before Starting The following items are supplied in the miRNA Amplification Module 10X miRNA Reaction Buffer 25 mM MnCl2 10 mM ATP Poly A Polymerase DEPC treated water The following items are supplied by the user Up to 30 ng of enriched miRNA per sample Optional Depending on the amount of purified sample a SpeedVac Concentrator Savant Instruments Inc or similar instrument may be necessary to concentrate the sample 1 mM Tris pH 8 0 Microcentrifuge Heat block or water bath set at 37 C 1 5 ml RNase free microcentrifuge tubes We do not recommend using the NCode Multi Species miRNA Microarray Controls in the following amplification procedure We recommend using these controls in the subsequent labeling procedure as described in the NCode miRNA Labeling System manual The following reaction uses the 10 mM ATP included in the kit not the 100 mM ATP using in the In Vitro Transcription reaction page 18 Be careful to select the vial of 10 mM ATP for use in the following reaction Continued on next page
21. e and discard the Spin Cartridge Do not discard the flow through 5 Add 400 l of 100 ethanol to the flow through and mix well by vortexing 6 Transfer 500 l of the sample from Step 5 to a new Spin Cartridge in a Collection Tube Procedure continued on next page Continued on next page 8 Isolating Small RNA continued Isolating Small RNA Using the PureLink miRNA Isolation Kit continued Procedure continued from previous page 7 Centrifuge the Spin Cartridge at 12 000 g for 1 minute at room temperature 8 Transfer the remaining sample 500 l from Step 5 to the Spin Cartridge from Step 6 and centrifuge at 12 000 g for 1 minute at room temperature 9 Discard the flow through and re insert the Spin Cartridge into the Collection Tube 10 Add 500 l of Wash Buffer W5 prepared with ethanol see above to the Spin Cartridge 11 Centrifuge 12 000 g for 1 minute at room temperature 12 Repeat Steps 10 11 one more time 13 Discard the flow through and place the Spin Cartridge into a Wash Tube supplied with the kit 14 Centrifuge the Spin Cartridge at maximum speed for 2 3 minutes at room temperature to remove any residual Wash Buffer Discard the Wash Tube 15 Place the Spin Cartridge into a clean 1 7 ml Recovery Tube supplied with the kit 16 Add 50 100 l of sterile RNase free water pH gt 7 0 supplied with the kit to the center of the Spin Cartridge higher e
22. efly centrifuge and then return to ice 2 In a separate RNase free tube add the following reagents and mix gently by hand Amounts are provided per reaction For multiple reactions prepare a master mix with a 5 10 overage to enable accurate pipetting Component Volume 10X miRNA Reaction buffer 2 l 10 mM dTTP 4 l Terminal Deoxynucleotidyl Transferase 2 l DEPC treated water 2 l 3 Add the 10 l reaction mix above to the cDNA from Step 1 for a final volume of 20 l Cap the tube mix gently by hand and briefly centrifuge 4 Incubate in a 37 C heat block for 3 minutes 5 Stop the reaction by heating at 80 C for 10 minutes Briefly centrifuge and cool to room temperature for 1 2 minutes Proceed immediately to T7 Promoter Synthesis next page 17 T7 Promoter Synthesis Introduction In this step you synthesize a T7 promoter on the poly dT tail of the cDNA using Klenow enzyme Before Starting The following items are supplied in the miRNA Amplification Module T7 Template Oligo Klenow 10 mM dNTP Mix 10X miRNA Reaction Buffer The following items are supplied by the user Heat block or thermal cycler at 37 C Ice Microcentrifuge The T7 Template Oligo includes a DNA polymerase blocker element that prevents complete second strand synthesis of the cDNA T7 Promoter Synthesis Procedure Perform the following synthesis reaction for each vial
23. ified 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 29 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 Greene Publishing Associates and Wiley Interscience New York Bentwich I Avniel A Karov Y Aharonov R Gilad S Barad O Barzilai A Einat P Einav U Meiri E Sharon E Spector Y and Bentwich Z 2005 Identification of hundreds of conserved and nonconserved human microRNAs Nature Genet 37 766 770 Gerard G F 1994 Inhibition of SuperScript II Reverse Transcriptase by Common Laboratory Chemicals FOCUS 16 102 103 Goff L A Yang M Bowers J Getts R C Padgett R W and Hart R P 2005 Rational pr
24. inued Procedure continued from the previous page 4 Add 500 l of Wash Buffer II prepared with ethanol see previous page to the spin cartridge Centrifuge at 12 000 g for 15 seconds at room temperature Discard the flow through and re insert the cartridge in the tube 5 Repeat Step 4 once 6 Centrifuge the spin cartridge at 12 000 g for 1 minute at room temperature to dry the membrane with attached senseRNA 7 Discard the collection tube and insert the cartridge into an RNA Recovery Tube supplied with the kit 8 To elute the senseRNA add 30 l of DEPC treated water to the center of the spin cartridge and incubate at room temperature for 1 minute 9 Centrifuge the spin cartridge for 2 minutes at 12 000 g at room temperature to collect the eluate The eluate contains your purified senseRNA Prior to fluorescent labeling calculate the yield of the purified senseRNA as described in Quantifying senseRNA on page 22 Alternatively store the sample at 80 C 22 Quantifying senseRNA Determining Yield Using an RNA Quantitation Kit We recommend using the Quant iT Ribogreen RNA Assay Kit or the RediPlate 96 Ribogreen RNA Quantitation Kit for highly sensitive quantitation of small amounts of RNA using a fluorescence microplate reader Ordering information is provided on page vi Each kit provides highly accurate fluorescent quantification of minute quantities of RNA in the range of 1
25. ity with endogenous miRNAs from model organisms and is provided at a concentration compatible with endogenous miRNA expression levels The NCode SYBR Green miRNA qRT PCR Kit provides qualified reagents for the detection and quantitation of miRNAs in real time quantitative RT PCR qRT PCR This kit has been optimized for the detection and quantification of miRNA from 10 ng to 2 5 g of total RNA using a SYBR Green detection platform The NCode SYBR GreenER miRNA qRT PCR Kit provides qualified reagents for the detection and quantitation of miRNAs in real time quantitative RT PCR qRT PCR This kit has been optimized for the detection and quantification of miRNA from 10 ng to 2 5 g of total RNA using a SYBR GreenER detection platform The NCode miRNA First Strand cDNA Synthesis Kit provides qualified reagents for the polyadenylation of miRNAs from total RNA and synthesis of first strand cDNA from the tailed miRNAs for use in real time quantitative PCR qPCR This kit has been optimized for the detection and quantification of miRNA from 10 ng to 2 5 g of total RNA using a SYBR Green or SYBR GreenER detection platform sold separately Continued on next page 4 Introduction continued NCode System Workflow Diagram Continued on next page 5 Introduction continued Materials Supplied by the User In addition to the kit components you should have the following items on hand
26. l T7 Enzyme Mix 7 l 5 Cap the tube mix gently by hand and briefly centrifuge 6 Incubate for 4 16 hours at 37 C in an air incubator do not use a heat block water bath or thermocycler see Important note on the previous page Following incubation the senseRNA may be stored at 80 C Otherwise proceed to Purification of the senseRNA page 20 20 Purifying senseRNA Introduction Following preparation of the senseRNA purify the sample according to the guidelines in this section PureLink Micro to Midi Total RNA Purification System We recommend using the PureLink Micro to Midi Total RNA Purification System for cleanup of senseRNA samples Invitrogen catalog no 12183 018 see page vi This kit has been extensively tested with the NCode miRNA Amplification System The PureLink Micro to Midi System uses a silica based membrane in a spin column format and can be used to purify high quality RNA from very small quantities of sample Other small sample RNA cleanup kits may also be appropriate for purification of senseRNA samples Purifying senseRNA using the PureLink Micro to Midi System The following protocol has been adapted from the Liquid Samples cleanup protocol in the PureLink Micro to Midi Total RNA Purification System manual Materials needed Components of the PureLink Micro to Midi Total RNA Purification System Invitrogen catalog no 12183 018
27. ldg 6F 3 9 15 Kaigan Minato ku Tokyo 108 0022 Tel 81 3 5730 6509 Fax 81 3 5730 6519 E mail jpinfo invitrogen com European Headquarters Invitrogen Ltd Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Tel 44 0 141 814 6100 Tech Fax 44 0 141 814 6117 E mail eurotech invitrogen com Material Data Safety Sheets MSDSs MSDSs are available on our Web site at www invitrogen com On the home page click on Technical Resources and follow instructions on the page to download the MSDS for your product Limited Warranty 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 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 spec
28. lution volumes may increase yield but will result in more dilute sample 17 Incubate at room temperature for 1 minute 18 Centrifuge the Spin Cartridge at maximum speed for 1 minute at room temperature 19 The Recovery Tube contains purified small RNA molecules Remove and discard the cartridge Note The recovery of the elution volume will vary and is usually 90 of the elution buffer volume used Store the purified product at 80 C or proceed to quantification as described in the next section 9 Quantifying Small RNA Introduction In this step you determine the quantity of isolated small RNA prior to polyadenylation This quantity is used to determine the amount of ATP to use in the polyadenylation procedure see next page Quantifying the Amount of Small RNA Isolated small RNA is typically too dilute to determine the quantity using A260 absorbance on a standard spectrophotometer We recommend using the Quant iT Ribogreen RNA Assay Kit or the RediPlate 96 Ribogreen RNA Quantitation Kit Ordering information is provided on page vi Each kit provides highly accurate fluorescent quantification of minute quantities of RNA in the range of 1 1 000 ng ml An undiluted sample of small RNA from the PureLink miRNA Isolation Kit should fall well within the linear range of the assay The assay takes approximately 1 hour to complete Alternatively a capillary or other small volume spectrophotometer may b
29. nt Volume 5X First Strand buffer 10 l 0 1 M DTT 5 l 10 mM dNTP Mix 2 5 l RNaseOUT 40 U l 1 l SuperScript III RT 200 U l 2 l DEPC treated water 2 5 l 6 Mix the tube gently by hand do not vortex and incubate at 46 C for 1 hour 7 Add 8 75 l of 0 5 M NaOH 50mM EDTA to stop the reaction Note that the reaction may turn to a brown color this is normal 8 Briefly vortex and centrifuge the tube to collect the contents 9 Incubate the tube at 65 C for 30 minutes to degrade the miRNA Note that the reaction may turn from brown to clear this is normal 10 Neutralize the reaction by adding 12 5 l of 1 M Tris pH 8 0 Briefly vortex and centrifuge the tube 11 Bring the reaction volume up to 100 l by adding 28 75 l of 1X TE buffer Proceed immediately to Purification and Concentration of cDNA next page 14 Purification and Concentration of First Strand cDNA Introduction In this step you purify and concentrate the first strand cDNA using the cDNA Ultrafiltration Columns and Tubes provided in the kit Before Starting The following items are supplied in the cDNA Purification Module cDNA Ultrafiltration Columns cDNA Ultrafiltration Tubes The following item is supplied in the miRNA Amplification Module DEPC treated water The following items are supplied by the user 1X TE buffer 10 mM Tris HCl 0 1 mM EDTA pH 8 0 10 mM Tris pH 8 0
30. obe optimization and enhanced detection strategy for microRNAs using microarrays RNA Biology 2 published online Imanishi T Itoh T Suzuki Y and O Donovan C 2004 Integrative annotation of 21 037 human genes validated by full length cDNA clones PLoS Biol 2 e162 John B Enright A J Aravin A Tuschl T Sander C and Marks D S 2004 Human MicroRNA Targets PLoS Biol 2 e363 Lagos Quintana M Rauhut R Lendeckel W and Tuschl T 2001 Identification of novel genes coding for small expressed RNAs Science 294 853 858 Lim L P Glasner M E Yekta S Burge C B Bartel D P 2003 Vertebrate microRNA Genes Science 299 1540 Nakahara K and Carthew R W 2004 Expanding roles for miRNAs and siRNAs in cell regulation Curr Opin Cell Biol 16 127 133 Okazaki Y Furuno M Kasukawa T and Adachi J 2002 Analysis of the mouse transcriptome based on functional annotation of 60 770 full length cDNAs Nature 420 563 573 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Ed Cold Spring Harbor Laboratory Press Plainview New York Stark A Brennecke J Russell R B and Cohen S M 2003 Identification of Drosophila MicroRNA Targets PLoS Biol 1 E60 Xie X Lu J Kulbokas E J Golub T R Mootha V Lindblad Toh K Lander E S and Kellis M 2005 Systematic discovery of regulatory motifs in human promoters and 3 UTRs by c
31. of developmental timing and pattern formation Lagos Quintana et al 2001 restriction of differentiation potential Nakahara amp Carthew 2004 regulation of insulin secretion Stark et al 2003 and genomic rearrangements John et al 2004 Several unique physical attributes of miRNAs including their small size lack of poly adenylated tails and tendency to bind their mRNA targets with imperfect sequence homology have made them elusive and challenging to study In addition strong conservation between miRNA family members means that any detection technology must be able to distinguish between 22 base sequences that differ by only 1 2 nucleotides Recent advances in spotted oligonucleotide microarray labeling and detection have enabled the use of this high throughout technology for miRNA screening Continued on next page 3 Introduction continued Other Products in the NCode System The NCode miRNA Amplification System was designed and developed in conjunction with the following products for ordering information see page vi The PureLink miRNA Isolation Kit is designed to purify small 200 nt cellular RNA molecules including regulatory RNA molecules such as miRNA and short interfering RNA siRNA The kit uses a silica based two column system to enrich small RNA from various sample sources including cells tissues and total RNA The enriched miRNA from this kit can be used directly in the NCode
32. of sample from Step 1 Component Volume Tube from Step 1 18 l 10X miRNA Reaction Buffer 2 5 l 25 mM MnCl2 2 5 l Diluted ATP from Step 2 1 l Poly A Polymerase 1 l Final Volume 25 l 4 Mix gently and centrifuge the tube briefly to collect the contents 5 Incubate the tube in a heat block or water bath at 37 C for 15 minutes After incubation proceed immediately to Reverse Transcription of Tailed miRNA next page 12 Reverse Transcription of Tailed miRNA Introduction After you have polyadenylated the miRNA you are ready to synthesize first strand cDNA from the tailed miRNA Before Starting The following items are supplied in the miRNA Amplification Module Oligo dT 24V primer 5X First Strand Buffer 0 1 M DTT 10 mM dNTP Mix RNaseOUT SuperScript III RT DEPC treated water The following items are supplied by the user Polyadenylated miRNA from previous procedure 0 1X TE buffer 1 mM Tris HCl 0 1 mM EDTA pH 8 0 1X TE buffer 10 mM Tris HCl 0 1 mM EDTA pH 8 0 0 5 M NaOH 50 mM EDTA 1 M Tris pH 8 0 Vortex mixer Incubator s thermal cycler s set at 46 C and 65 C 1 5 ml RNase free microcentrifuge tubes Ice RNaseOUT Recombinant RNase Inhibitor RNaseOUT Recombinant RNase Inhibitor has been included in the system to safeguard against degradation of target RNA due to
33. of tailed cDNA 1 Add 2 l of T7 Template Oligo to the tailed cDNA from Step 5 page 16 for a volume of 22 l 2 Incubate at 37 C for 10 minutes to anneal the strands 3 To each reaction tube add the following components for a final reaction volume of 25 l For multiple reactions you can prepare a master mix of the following to enable accurate pipetting Component Volume 10X miRNA Reaction buffer 1 l 10 mM dNTP Mix 1 l Klenow 1 l 4 Cap the tube mix gently and briefly centrifuge Incubate at room temperature for 30 minutes 5 Stop the reaction by heating at 65 C for 10 minutes and then place on ice Proceed immediately to In Vitro Transcription next page 18 In Vitro Transcription Introduction In this step you generate senseRNA from the first strand cDNA using T7 RNA Polymerase in a proprietary enzyme mix Before Starting The following items are supplied in the miRNA Amplification Module T7 Enzyme Mix 10X T7 Reaction Buffer 100 mM ATP 100 mM CTP 100 mM GTP 100 mM UTP The following items are supplied by the user Microcentrifuge Vortex mixer Air incubator set at 37 C see Important note below Longer in vitro transcription incubation times will result in higher yields For optimal results we recommend performing an overnight incubation and proceeding with purification quantitation and labeling of the senseRNA on the following
34. omparison of several mammals Nature 434 338 345 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use User Manual 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 speci c contact information visit our web site at www invitrogen com
35. quivalent cells or tissue For smaller amounts of starting material you can use the cDNA Ultrafiltration Columns and Tubes provided in this kit or Microcon YM 100 columns and tubes from Millipore as described in the protocol starting on page 25 Using the PureLink kit 300 500 ng of total RNA or equivalent cells or tissue typically yields 15 30 ng of small RNA molecules depending on the sample PureLink miRNA Isolation Kit The NCode miRNA Amplification System was developed and optimized using enriched miRNA from the PureLink miRNA Isolation Kit Invitrogen catalog no K1570 01 see page vi The PureLink kit provides columns reagents and protocols for isolating small RNA molecules from a variety of cell and tissue types in small and large sample volumes The PureLink kit may be used to isolate small RNA from 300 500 ng of total RNA or equivalent cells or tissue Continued on next page 7 Isolating Small RNA continued When using the PureLink miRNA Isolation Kit to isolate small RNA use the protocol on the following pages which has been adapted from the standard PureLink protocol Note that the following protocol uses 100 ethanol which removes more debris from the sample enhancing the performance of this kit We recommend starting with high quality isolated small RNA We do not recommend using total RNA for amplification labeling and hybridization The quality of the RNA is c
36. rect compatibility with miRNA probe sequences on microarrays Amplified senseRNA is ideal for expression profiling from very small amounts of starting material because it preserves the relative abundance of the different miRNA sequences in the original sample allowing you to compare relative quantities across experiments This system is designed for use with up to 30 ng of miRNA isolated from 300 500 ng of total RNA as starting material The procedure allows for isolation and preparation of the sample in a single day followed by in vitro transcription with an overnight incubation The following day you are ready to purify and label your amplified senseRNA population for downstream analysis Experimental Outline Continued on next page 2 Introduction continued System Overview After isolating small RNA from cells tissue or total RNA with the PureLink miRNA Isolation Kit use the NCode miRNA Amplification System to amplify the enriched miRNAs as described below First you add a poly A tail to the miRNA using poly A polymerase and an optimized reaction buffer Then you reverse transcribe the tailed miRNA using SuperScript III RT and purify and concentrate the resulting first strand cDNA Next you add a poly dT tail to the 3 end of the first strand product using terminal deoxynucleotidyl transferase and synthesize and anneal a T7 promoter on the tailed cDNA using Klenow enzyme and a specially
37. ribonuclease contamination of the RNA preparation Diluting Oligo dT 24V Primer The Oligo dT 24V Primer provided in the kit must be diluted 1 10 in 0 1X TE buffer before use We recommend diluting 10 l of the Oligo dT 24V in 90 l of 0 1X TE buffer and preparing 20 5 l single use aliquots to minimize free thaw cycles Note that each kit contains 15 l of undiluted oligo You will need only 2 l of the diluted Oligo dT 24V per cDNA synthesis reaction Continued on next page 13 Reverse Transcription of Tailed miRNA continued First Strand cDNA Synthesis The following procedure is for a single reaction For multiple reactions prepare a master mix of the RT reaction mix with a 5 10 overage to enable accurate pipetting 1 Briefly centrifuge the 25 l of polyadenylated miRNA from Step 5 page 11 and place on ice 2 If you haven t already done so prepare a 1 10 dilution of Oligo dT 24V primer as described on the previous page Vortex and briefly centrifuge 3 Add 2 l of diluted Oligo dT 24V primer to the tube of miRNA on ice Mix and briefly centrifuge 4 Incubate at 65 C for 10 minutes and then immediately transfer the tube to ice for 2 minutes 5 Briefly vortex and centrifuge each of the following reagents and then add them to the tube on ice for a final reaction volume of 50 l For multiple reactions prepare a master mix with a 5 10 overage to enable accurate pipetting Compone
38. ritical for amplification In amplification labeling and array hybridization applications the presence of contaminants in the RNA may reduce amplification yield and increase background fluorescence in microarrays Carefully follow the recommendations below to prevent contamination Isolating Small RNA Using the PureLink miRNA Isolation Kit The following protocol has been adapted from the PureLink miRNA Isolation Kit manual See that manual for more details The following protocol may be used to isolate up to 30 ng of miRNA from 300 500 ng of total RNA Materials needed Components of the PureLink miRNA Isolation Kit Invitrogen catalog no K1570 01 see page vi Total RNA sample 100 ethanol Microcentrifuge RNase free pipette tips Wash Buffer W5 Prepare Wash Buffer W5 for use by adding 40 ml of 96 100 ethanol to 10 ml of Wash Buffer W5 included with the kit Store the Wash Buffer W5 with ethanol at room temperature Procedure 1 Resuspend total RNA in 300 l of Binding Buffer L3 supplied with the PureLink kit Mix well by vortexing or pipetting up and down 2 Add 300 l of 100 ethanol to the solution Mix well by vortexing 3 Add the complete solution 600 l to a Spin Cartridge preinserted in a Collection Tube from the PureLink kit 4 Centrifuge the Spin Cartridge at 12 000 g for 1 minute at room temperature to collect the flow through Remov
39. see page vi 2 mercaptoethanol 96 100 ethanol Microcentrifuge 1 5 ml RNase free microcentrifuge tubes RNase free pipette tips RNA Lysis Solution Prepare the RNA Lysis Solution included with the system fresh for each use by adding 1 v v 2 mercaptoethanol e g add 10 l of 2 mercaptoethanol to every 1 ml of RNA Lysis Solution Use 1 volume of freshly prepared RNA Lysis Solution for each volume of liquid sample Wash Buffer II Before using the Wash Buffer II included with the system for the first time add 60 ml of 96 100 ethanol directly to the bottle Check the box on the Wash Buffer II label to indicate that ethanol was added Procedure 1 To one volume of liquid sample e g 42 l of senseRNA from Step 5 page 19 add one volume of RNA Lysis Solution prepared with 2 mercaptoethanol see above followed by the same volume of 96 100 ethanol e g to 42 l of senseRNA add 42 l of RNA Lysis Solution followed by 42 l of ethanol 2 Mix by vortexing or pipetting up and down 5 times 3 Pipette the sample onto the RNA Spin Cartridge and centrifuge at 12 000 g for 15 seconds at room temperature Remove the cartridge from the tube discard the flow through and re insert the cartridge in the tube Procedure continued on the next page Continued on next page 21 Purifying senseRNA continued Purifying senseRNA using the PureLink Micro to Midi System cont
40. you choose to use Millipore s Microcon YM 100 Centrifugal Filters be careful to follow the procedure on the next page not the manufacturer s protocol provided with the columns The following procedure differs from the manufacturer s protocol and has been optimized for use with this kit Continued on next page 26 Isolating Small Amounts of Small RNA continued Isolation Procedure Use the following procedure to isolate small RNA from small amounts of total RNA 1 Insert the filtration column into the specially designed snap top tube You will need a separate column and tube for each sample processed 2 Pipette 50 500 g of total RNA in a volume of 70 l DEPC treated water onto the membrane in the center of the column Do not touch the membrane with the pipette tip 3 Secure the tube cap and insert the assembly in a centrifuge Note Align the cap strap toward the center of the rotor and be sure to counterbalance the rotor with a similar device 4 Centrifuge for 6 minutes at 13 000 g The eluate collected in the tube is your isolated small RNA Proceed to Quantifying Small RNA on page 9 Assembled column tube Unassembled column tube 27 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology 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
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