NCode™ miRNA Labeling System
Contents
1. Follow the guidelines on page 6 to prevent RNase contamination of the RNA preparation Always use fresh samples or samples frozen at 80 C If you are starting with total RNA analyze it by agarose ethidium bromide gel electrophoresis prior to isolation of small RNA Make sure that the chamber is properly sealed with the correct amount of liquid prior to incubation See the volumes specified in the Tagged miRNA Hybridization Procedure on page 16 Make sure that the Hybridization Mix completely covers the array surface under the LifterSlip coverslip Avoid direct exposure of the Alexa Fluor 3 and Alexa Fluor 5 Capture Reagents and the hybridized array to light Perform hybridization and wash procedures in low light conditions Check the temperatures of all incubators with a calibrated thermometer Follow the guidelines on page 6 to prevent RNase contamination of the RNA preparation Always use fresh samples or samples frozen at 80 C If you are starting with total RNA analyze it by agarose ethidium bromide gel electrophoresis prior to isolation of small RNA If you are printing your own arrays carefully follow all array blocking procedures NCode Multi Species miRNA Microarrays come fully blocked and ready to use Array slide scanned in Check the position of the slide in the scanner wrong orientation reposition and rescan if necessary Continued on next page Troubleshooting continued High or uneven Resid2. A typical lower limit of detection LLD is 8 times the median local background of all array features The signal background S B ratio is calculated by dividing the median signal of positive features by the median background Be careful to position the slide in the proper orientation in the microarray scanner If no signal is apparent after scanning double check the orientation of the slide Consult your scanner documentation for details When scanning dual color arrays we recommend examining the image histogram available with GenePix Pro software to determine whether the signal intensities in the two channels are comparable 25 Appendix Troubleshooting Yield of enriched miRNA is low Coverslip stuck to array surface Low or no overall fluorescent signal intensity on the array 26 Problems with the small RNA isolation procedure Degraded starting material Hybridization chamber not properly sealed or humidified Inadequate volume of Hybridization Mix used for coverslip size Photobleaching of the Alexa Fluor Capture Reagents Incubation temperatures during hybridization were incorrect Degraded starting material Insufficient blocking of the array prior to hybridization 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
3. Alexa Fluor 5 Capture Reagents rer Alexa Fluor Capture Reagent Day 2 marsis miRNA probe Continued on next page Overview continued MicroRNAs Advantages of the System Alexa Fluor Labeling Technology 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 of target mRNAs by binding to partially complementary sites in the 3 untranslated regions UTRs of the message Lim 2003 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 little is known about their cellular function They have been implicated in regulation 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 miRN As 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 dete
4. Mix gently and then centrifuge the tube briefly to collect the contents Incubate at room temperature 20 28 C for 30 minutes 5 Stop the reaction by adding 4 ul of 0 5 M EDTA at room temperature Briefly vortex and then centrifuge the tube 6 Add 60 pl of 1X TE Buffer at room temperature for a final volume of 100 ul Briefly vortex and then centrifuge the tube Proceed to Purifying the Tagged miRNA next page Purifying the Tagged miRNA Introduction Note Before Starting Purification Procedure In this step you purify the tagged miRNA e If you are co hybridizing two samples on the same array the tagged miRNA from both samples may be loaded onto the same purification column Be careful to load each sample separately as described below Do not mix the samples before loading to prevent cross ligation e During purification you can begin thawing the 2X Hybridization Solution for the next procedure see Step 1 Tagged miRNA Hybridization Procedure page 16 The following items are supplied in the NCode miRNA Purification Module e Low Elution Volume Spin Cartridges preinserted into collection tubes e Collection Tubes e Binding Buffer prepared with isopropanol as described on page vi e Wash Buffer prepared with ethanol as described on page vi e DEPC treated water The following items are supplied by the user e Microcentrifuge e Vortex mixer Use the following procedure to purify the tagged miRNA using
5. The elution volume from the PureLink miRNA Isolation Kit is 50 100 ul Optional Add 1 pl of diluted NCode Multi Species miRNA Microarray Controls to each tube of sample see the product insert provided with the controls for the dilution procedure Note that the concentration of the controls is so low that you do not need to factor it into the amount of enriched miRNA for the calculation in Step 3 below Note If you are not using the controls add DEPC treated water to each tube to a final volume of 15 5 ul Based on the quantity of enriched miRNA 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 200 ng of miRNA the ATP dilution factor is 5000 200 ng 25 Dilute the ATP 1 25 by adding 1 ul of 10 mM ATP to 24 ul of 1 mM Tris pH 8 0 2 Add the following at room temperature to the tube of sample from Step 2 Component Volume Tube from Step 2 155 pl 5X miRNA Reaction Buffer 5yl 25 mM MnCl 2 5 pl Diluted ATP from Step 3 above 1 ul Poly A Polymerase ul Final Volume 25 ul 3 Mix gently and then centrifuge the tube briefly to collect the contents 4 Incubate the tube at 37 C for 15 minutes After incubation proceed immediately to Ligation of the Capture Sequence next page 11 Ligation of the Capture Seguence Introduction Before Starting 6X Alexa Fluor Ligation Mix Ligation Proce
6. Prepare Wash Solutions Note Important Following the 4 hour hybridization of the Alexa Fluor Capture Reagents perform the final array wash procedure as described in this section The following items are supplied by the user e Wash Solution 1 2X SSC 0 2 SDS may be prepared from UltraPure 20X SSC and UltraPure 10 SDS Solution available from Invitrogen see page vii e Wash Solution 2 2X SSC e Wash Solution 3 0 2X SSC e Clean slide rack e 3clean wash containers capable of completely submerging an array slide in a slide rack e g 400 ml e An additional wash container or squirt bottle for washing off the coverslip e Incubator or water bath at 60 C e Vortex mixer e Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack or with a slide holder Prepare appropriate volumes of Wash Solution 1 2X SSC 0 2 SDS Wash Solution 2 2X SSC and Wash Solution 3 0 2X SSC prior to beginning the wash procedure Prepare enough of each solution to fully submerge the array slide in a slide rack in a wash container filled with the solution The table below provides volumes for preparing 2 liters of each solution from UltraPure 20X SSC UltraPure 10 SDS and dH 0 Wash Solution 1 Wash Solution 2 Wash Solution 3 UltraPure 20X SSC 200 ml 200 ml 20 ml UltraPure 10 SDS 40 ml dH 0 to 2 liters to 2 liters to 2 liters The following procedure has been designed for
7. 900 Alexa Fluor molecules as well as highly specific sequences complementary to the ligated tags on the hybridized mi RNAs Note that Alexa Fluor 3 is identical to Alexa Fluor 546 and Alexa Fluor 5 is identical to Alexa Fluor 647 Excitation and emission maxima are listed below Excitation Emission Alexa Fluor 3 556 nm 573 nm Alexa Fluor 5 650 nm 665 nm Capture reagents contain 3DNA reagent manufactured under license from Genisphere Inc Continued on next page 20 Hybridization of Alexa Flour Capture Reagents continued Important Preparing the Alexa Fluor Capture Reagents Hybridization of Alexa Fluor Capture Reagents Procedure During the following procedure minimize exposure of Alexa Fluor Capture Reagents and the hybridized array to direct light to avoid photobleaching Always wear powder free latex gloves when handling arrays Avoid contact with the printed array surface The array surface should remain as lint free and dust free as possible Before hybridization prepare the Alexa Fluor Capture Reagents as follows 1 Thaw the Alexa Fluor 3 and or Alexa Fluor 5 Capture Reagents in the dark at room temperature for 20 minutes Vortex the tube s at maximum speed for 3 seconds then centrifuge briefly Incubate in the dark at 50 55 C for 10 minutes Vortex the tube s at maximum speed for 3 4 seconds and then centrifuge briefly to collect the contents Follow the protocol below to h
8. Ribogreen RNA Quantitation Kit see page vii TM Recommended NCode Multi Species miRNA Microarray see page vii or TM NCode miRNA Microarray Probe Sets see page vii printed on epoxy coated glass slides e g Corning Epoxide Coated Slides catalog 40041 or 40044 Recommended NCode Multi Species miRNA Microarray Controls see page vii Hybridization chamber e g Corning Hybridization Chamber catalog 2551 or 40080 Raised edge coverslips for the NCode Microarray we recommend LifterSlips Erie Scientific catalog 25x601 2 4789 Lint free laboratory wipes Slide rack 3 clean wash containers capable of completely submerging an array slide in a slide rack e g 400 ml Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack or with a slide holder Digital microarray scanner e g the GenePix 4000B from Molecular Devices and associated software Incubators water baths Vortex mixer SpeedVac Concentrator Savant Instruments Inc or similar concentrator Microcentrifuge Aerosol resistant pipette tips 1 5 ml RNase free microcentrifuge tubes 1 mM Tris pH 8 0 0 5 M EDTA 1X TE Buffer 2X SSC 0 2 SDS may be prepared from 20X SSC and 10 SDS available from Invitrogen see page vii 2X SSC 0 2X SSC 100 Isopropanol 100 Ethanol Methods Isolating Small RNA Introduction Note General Handling of RNA PureLink miRNA Isolat
9. System TM The NCode miRNA Labeling System was designed and developed in conjunction with the following products for ordering information see page vii The PureLink miRNA Isolation Kit is designed to purify small lt 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 miRNA Labeling System TM The NCode miRNA Amplification System is a robust system for amplifying senseRNA molecules from minute guantities lt 30 ng of miRNA The system provides consistent and accurate 21000 fold amplification while preserving the relative abundance of the miRNA sequences in the original sample allowing you to compare relative quantities across experiments The resulting amplified miRNA is in the sense orientation for direct TM compatibility with NCode microarrays and probe sequences 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 mikBase 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 h
10. is a registered trademark of Savant Instruments Inc LifterSlip is a trademark of Erie Scientific Company Technical Service Web Resources Visit the Invitrogen Web site 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 service 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 listed on our Web page 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 serviceGinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com Material Data Safety Sheets MSDSs Limited Warranty 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 f
11. isopropanol indicated below directly to each 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 Amount Binding Buffer 18 ml entire bottle 100 Isopropanol 6 5 ml Final Volume 24 5 ml The Binding Buffer plus isopropanol is stable for at least six months at room temperature Each bottle of Wash Buffer must be mixed with 100 ethanol prior to use Add the amount of ethanol indicated below directly to each 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 Amount Wash Buffer 5 ml entire bottle 10096 Ethanol 20 ml Final Volume 25 ml The Wash Buffer plus ethanol is stable for at least six months at room temperature The Certificate of Analysis CofA provides detailed guality control information for this product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number Related Products Additional The NCode miRNA Labeling System is part of an integrated microRNA Products expression profiling system that includes isolation 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 29 N
12. ome tien ete e ee ead 30 iii iv Kit Contents and Storage TM Shipping and The NCode miRNA Labeling System is shipped in two modules The Labeling Storage Module is shipped on dry ice while the Purification Module is shipped at room temperature Store the components of the Labeling Module at 20 C and the components of the Purification Module at room temperature NCode miRNA Components should be stored at 20 C Reagents are provided for 20 labeling Labeling Module and hybridization reactions 10 mM ATP Poly A Polymerase DEPC treated water T4 DNA Ligase 1 U ul 2X Hybridization Solution 6X Alexa Fluor 3 Ligation Mix 6X Alexa Fluor 5 Ligation Mix NCode miRNA Components should be stored at room temperature Purification Module Low Elution Volume Spin Cartridges with collection tubes 2 x 11 columns Binding Buffer must be combined with 6 5 ml of 100 isopropanol 18 ml to create final buffer see next page Wash Buffer must be combined with 20 ml of 100 ethanol to 5 ml create final buffer see next page Collection Tubes Continued on next page TM Capture reagents contain 3DNA reagent manufactured under license from Genisphere Inc Kit Contents and Storage continued Preparing Binding Buffer with Isopropanol Preparing Wash Buffer with Ethanol Product Qualification vi Each bottle of Binding Buffer must be mixed with 10076 isopropanol prior to use Add the amount of
13. the 2X Hybridization Solution by heating it at 70 C for 10 minutes and then vortexing to resuspend evenly If necessary repeat heating and vortexing until the buffer is fully resuspended 2 The amount of Hybridization Mix per array depends on the coverslip size Prepare as follows Amount per Array Component 24 x 50 Coverslip 25 x 60 Coverslip Tagged miRNA from Purification Procedure Step 10 page 14 20 ul 20 ul DEPC treated water 5 pl 2X Hybridization Solution 20 ul 25 ul Final Volume 40 ul 50 ul 3 Gently vortex and briefly centrifuge the Hybridization Mix then incubate the mix at 75 80 C for 10 minutes Hold the mix at the hybridization temperature 52 C until loading the array Protocol continued on next page Continued on next page Tagged miRNA Hybridization continued Tagged miRNA Protocol continued from previous page Hybridization 4 Using powder free latex gloves inspect the coverslip to ensure it is clean If Procedure necessary gently wipe clean with a lint free laboratory wipe continued 5 Place the slide with the array facing up in an open clean dry hybridization chamber The array on the NCode slide is printed on the same side as the barcode The NCode microarray comes blocked and ready to use 6 Gently vortex and briefly centrifuge the Hybridization Mix Then e ForLifterSlips you may first place the slip on the array with the dull side of the white strips facing down along
14. the components of the Purification Module 1 Add 700 pl Binding Buffer prepared with isopropanol to each tube of labeled miRNA from Ligation Procedure Step 6 page 12 Mix thoroughly by vortexing and centrifuge briefly to collect the contents 2 Each Low Elution Volume Spin Cartridge is preinserted into a collection tube Load the entire volume of miRNA Binding Buffer from a single tube directly onto the Spin Cartridge If you have two samples for co hybridization load only one sample at this time 3 Centrifuge the Spin Cartridge at 3 300 x g in a microcentrifuge for 1 minute or until the entire volume passes through the cartridge Remove the collection tube and discard the flow through 4 Optional If you are co hybridizing two samples place the Spin Cartridge in the same collection tube and load the second sample on the cartridge Then repeat Step 3 5 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 Procedure continued on the next page Continued on next page 13 Purifying the Tagged miRNA continued Purification Procedure continued 14 6 10 Procedure continued from the previous page Centrifuge at maximum speed for 30 60 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 60 seconds to remove any re
15. Code Multi Species miRNA Microarray Control V2 MIRAC2 01 NCode Multi Species miRNA Microarray Probe Set V2 3 x 384 well plates MIRMPS2 01 500 pmol per well Quant iT Ribogreen RNA Assay Kit 200 2 000 cuvette assays R 11490 NCode SYBR Green miRNA qRT PCR Kit 10 polyadenylation MIRQ 100 20 cDNA synthesis 100 qPCR reactions NCode SYBR GreenER miRNA qRT PCR Kit 10 polyadenylation MIROER 100 20 cDNA synthesis 100 gPCR reactions NCode miRNA First Strand cDNA Synthesis Kit 10 polyadenylation MIRC 10 20 cDNA synthesis 50 polyadenylation MIRC 50 100 cDNA synthesis RediPlate 96 Ribogreen RNA Quantitation Kit 96 well plate 8 x 12 strip R 32700 wells UltraPure 20X SSC 1 liter 15557 044 4 liters 15557 036 UltraPure 10 SDS Solution 4 x 100 ml 15553 027 1 liter 24730 020 vii viii Overview Introduction System Overview Experimental Outline The NCode miRNA Labeling System is a robust and efficient system for labeling enriched microRNAs miRNAs with fluorescent Alexa Fluor dyes and hybridizing them to microarrays printed with species specific antisense miRNA probes The reagents in this kit have been optimized to ensure sensitive labeling from minimal RNA input enabling simple and efficient profiling of miRNA expression patterns in various types of tissue disease and developmental states TM First you isolate small RNA using the PureLink miRNA Isolation Kit or your metho
16. 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 comparison of several mammals Nature 434 338 345 2005 2007 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 30 invitrogen Corporate Headquarters 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
17. Invitrogen NCode miRNA Labeling System For generating labeled microRNA molecules for hybridization to microarrays Catalog no MIRLS 20 Version D 17 September 2007 25 0888 ii Table of Contents Kit Contents and Storage iieb aena re igit ed end Ate da perde ied trenes v Related Products mummoa nm seien erneuert RB RR UE eet nae vii OVeEr VIG Wiima teeta uii dli iet od esed tese Poe Eesti ss 1 Quir m E a vn TO ta TE Ha an a 6 Isolating Small RNA a nn ere e os utc a cheney PONES RR SEN OSUTA Un rue 6 Quantifying Small RNA need utate tieu Rake norte e TT Ake ohh esie neer Aan fen lien 9 Polyadenylation of tiRNA tee eee tei ie te eerie ie ae kin ere e be RR END 10 Ligation of the Capture Sequence ssssssssssssseeeeeeenenenenenenrnrt a a a eee e eee 12 Purifying the Tagged miRNA nennen seen aped c pte abend i a loire 13 Tagged miRNA Hybridization es itoni eiee e a aeniea areke aia eare eaaa ees ebeit ioeie 15 Array WASH E se niece TA AT e ea E t enm eee E Rem 18 Hybridization of Alexa Flour Capture Reagents iie one vv u u 20 Firial Array Wash esae a ete Ee RH RE Ue a eee s 23 Array Imagine anid Analysis tte n eio tetuer tan einen Bi 25 PN 0 0121 ene 26 Trouble shoObng x5 tete sehen sales Bun am ie e PO eres AL SA Eek pa tele 26 Purchaser NotificatiOni ek ee ndi ecd aede ete o nitide di ed dette his 28 Technical Service snipee ora oe d eue MAAALA A aa RA TEA m EAA aa 29 References senden lhe
18. alyses Xie et al 2005 Probes are printed in duplicate Each NCode microarray comes blocked and ready to use and is provided in a kit of five slides See the user manual provided with the microarrays for more information including download instructions for the array GAL file e NCode miRNA Microarray Probe Sets are mammalian and non mammalian probe sets in 384 well plates dried down at 500 pmol per well See the product insert provided with the probe sets for resuspension and printing guidelines We have found that increasing the recommended hybridization temperature of 52 C by a few degrees to 54 56 C will increase hybridization specificity but reduce signal sensitivity We have found that 52 C provides an optimal balance TM of sensitivity and specificity with the NCode Multi Species miRNA Microarray Make sure that the thermometer on your hybridization incubator is properly calibrated The following procedure has been designed for use with epoxy coated glass slide microarrays If you are using a different type of array use hybridization solutions and protocols appropriate for that array TM e NCode miRNA microarray products are designed for coverslip hybridizations with volumes of 80 ul or less e Always wear powder free latex gloves when handling microarrays and coverslips Avoid contact with the printed array surface The array surface should remain as lint free and dust free as possible Open the s
19. capillary or other small volume spectrophotometer may be used for quantitation After quantifying the small RNA we recommend that you proceed directly to Poly A Tailing of miRNA on page 10 The RNA may be stored at 80 C if necessary Typical yields of small RNA molecules from various samples using the PureLink miRNA Isolation Kit are listed below The quantities in the table were determined using the Quant iT Ribogreen RNA Assay Kit see above Material Amount Yield HeLa cells 1 x 105 129 ug 293F cells 1 x 105 1 95 ug Mouse liver 5 mg 710 ng Rat Spleen 5 mg 1 39 ug Spinach 60 mg 1 64 ug Yeast S cerevisiae 1 x 106 6 21 ug Bacteria E coli 2 x 106 550 ng Polyadenylation of miRNA Introduction Important Before Starting NCode Multi Species miRNA Microarray Controls 10 After you have quantified the enriched miRNA you are ready to add a poly A tail to the miRNA TM If you are using amplified miRNA from the NCode miRNA Amplification System it is already polyadenylated Skip the following procedure and proceed directly to Ligation of the Capture Sequence on page 12 The following items are supplied in the NCode miRNA Labeling Module e 5X miRNA Reaction Buffer e 25mMMnCl e 10mMATP e Poly A Polymerase e DEPC treated water The following items are supplied by the user e Enriched miRNA from 10 ug of total RNA or equivalent cells or tissue per sample e SpeedVac Con
20. centrator Savant Instruments Inc or similar instrument e Optional NCode Multi Species miRNA Microarray Controls ordering information on page vii e 1mM Tris pH 8 0 e Microcentrifuge e Incubator or water bath set at 37 C e 15 ml RNase free microcentrifuge tubes NCode Multi Species miRNA Microarray Controls are 10 unique 20 22 nucleotide miRNA sequences that have been designed and screened as positive controls for use with NCode system These control sequences have been tested for cross reactivity with endogenous miRNAs from model organisms and are provided at a concentration compatible with endogenous miRNA expression levels NCode controls are provided at a concentration of 200 fmol yl and should be diluted 1 10 in DEPC treated water and stored in single use aliquots upon receipt as described in the product insert provided with the controls Add 1 ul of diluted control per sample as described in the procedure on the next page Continued on next page Polyadenylation of miRNA continued Polyadenylation Procedure Use the following procedure to add poly A tails to the enriched miRNA TM Note If you are using amplified miRNA from the NCode miRNA Amplification System skip this procedure and proceed directly to Ligation of the Capture Seguence on page 12 1 Following guantification of the enriched miRNA concentrate the sample in a SpeedVac Concentrator at low heat to a final volume of 14 5 ul Note
21. ction technology must be able to distinguish between 22 base sequences that differ by only 1 2 nucleotides Recent advances in microarray and gPCR detection have enabled the use of these technologies for miRNA screening TM The NCode miRNA Labeling System provides the following advantages e Requires less starting sample than comparable systems e Enriched miRNA samples can be tagged and hybridized in about an hour followed by an overnight incubation e Optimized reagents and protocol ensure highly robust and reproducible reactions TM e Designed and developed as part of the comprehensive NCode system which includes the NCode Multi Species miRNA Microarray Probe Sets Amplification System and Controls Alexa Fluor 3 and Alexa Fluor 5 Capture Reagents contain branched DNA polymers dendrimers each with a core that consists of a matrix of double stranded DNA and an outer surface comprised of hundreds of singled stranded arms The surface arms carry 900 Alexa Fluor molecules as well as highly specific sequences complementary to the ligated tags on the hybridized mi RNAs The high specificity of the binding sequences and high fluorescence of the Alexa Fluor dye molecules ensure maximum signal to background ratios and strong signal correlations Capture reagents contain 3DNA reagent manufactured under license from Genisphere Inc Continued on next page Overview continued Other Products in the NCode
22. d of choice and quantify If necessary amplify the enriched miRNA using the NCode miRNA Amplification System Then use the NCode miRNA Labeling System to tag the miRNAs in a simple 1 hour procedure and hybridize them to the microarray Using the Labeling System first you add a poly A tail to the miRNA using poly A polymerase and an optimized reaction buffer this step if unnecessary if you are using amplified miRNA Then you ligate a short highly specific tag sequence to each tailed miRNA using a bridging oligo Following a purification step you hybridize the tagged miRNA to the microarray and incubate overnight The next day you wash the array hybridize with Alexa Fluor 3 and Alexa Fluor 5 capture reagents perform another wash and scan using a standard microarray scanner The capture reagents are comprised of DNA polymers each with 900 Alexa Fluor molecules bound to a sequence complementary to the ligated tags on the hybridized miRNAs The high specificity of the binding sequence and high fluorescence of the dye molecules ensure maximum signal to background ratios and strong signal correlations 5 3 miRNA l Poly A tailing reaction 5 EE 3 Ligation of capture sequence to poly A tail Capture sequence Day 1 71 Hour Purification and hybridization of tagged miRNA to microarray Tagged miRNA 1150 Overnight miRNA probe Hybridization Wash and hybridization with Alexa Fluor 3
23. dure 12 Inthis step you ligate the capture seguence to the poly A tailed miRNA The following items are supplied in the NCode miRNA Labeling Module e 6X Alexa Fluor 3 Ligation Mix and 6X Alexa Fluor 5 Ligation Mix e T4 DNA Ligase e DEPC treated water The following items are supplied by the user e Polyadenylated miRNA from the previous page or polyadenylated amplified miRNA from the NCode miRNA Amplification System e Microcentrifuge e 05MEDTA e 1XTE Buffer Each 6X Alexa Fluor Ligation Mix includes two oligonucleotides e A31 base oligonucleotide tag complementary to the capture sequence in the Alexa Fluor Capture Reagent e A 19 base bridging oligo consisting of 9 bases complementary to the tag and 10 bases complementary to the poly A tail on each miRNA miRNA sequence AAAAAAAAAA 31 base Oligo Tag 19 base Bridging Oligo Use the following procedure to ligate the capture sequence to the tailed miRNA 1 Briefly centrifuge each tube containing 25 ul of poly A tailed miRNA from Poly A Tailing Procedure Step 6 previous page 2 Add the following components to each tube at room temperature If you will be co hybridizing two samples to the array use Alexa Fluor 3 Ligation Mix for one tube and Alexa Fluor 5 Ligation Mix for the other Component Volume Tube from Step 1 25 ul Alexa Fluor 3 Ligation Mix or Alexa Fluor 5 Ligation Mix 6 pl T4 DNA Ligase 2 pl DEPC Treated Water _3ul Final volume 36 ul
24. e sure the slide is completely submerged in the solution Do not expose the slide to air for more than a few seconds to avoid air drying 5 Wash the array in Wash Solution 1 for 10 15 minutes at room temperature with gentle agitation 150 200 rpm 6 Filla clean wash container with Wash Solution 2 at room temperature Transfer the slide rack with the slide to this wash container and wash with gentle agitation at room temperature for 10 15 minutes Do not expose the slide to air for more than a few seconds and make sure that the slide is completely submerged in Wash Solution 2 7 Filla clean wash container with Wash Solution 3 at room temperature Transfer the slide rack with the slide to this wash container and wash with gentle agitation at room temperature for 10 15 minutes Do not expose the slide to air for more than a few seconds and make sure that the slide is completely submerged in Wash Solution 3 Important Perform the next step quickly To avoid high background on the array do not allow the array surface to air dry prior to centrifugation 8 Prepare a centrifuge with a microtiter plate rotor adapter that will accept the slide rack containing the array slide Balance the opposing arm of the rotor with a slide rack containing an equivalent number of empty slides Quickly transfer the slide rack with the slide to the centrifuge and immediately spin for 2 4 minutes at 600 x g to dry Do not centrifuge at higher speeds or the s
25. g arm of the rotor with an slide rack containing an equivalent number of empty slides Quickly transfer the slide rack with the slide to the centrifuge and immediately spin for 2 4 minutes at 600 x g to dry Do not centrifuge at higher speeds or the slide might break Scan the array within hour after the final wash step to avoid photobleaching See Array Imaging and Analysis next page Array Imaging and Analysis Introduction Scanning the Microarray Note Following the final wash step arrays should be scanned immediately to minimize photobleaching of the Alexa Fluor dyes The array should be shielded from direct light and scanned within hour of completion the final wash to minimize photobleaching The NCode Multi Species miRNA Microarray may be scanned using a standard digital microarray scanner We recommend a scanner with a bit depth of at least 16 bits pixel The GenePix 4000B Molecular Devices is a common microarray scanner and includes software for analyzing the scanned image Alexa Fluor 3 and 5 have excitation and emission maxima identical to Alexa Fluor 546 and 647 respectively Program your scanner accordingly Excitation Emission Alexa Fluor 3 556 nm 573 nm Alexa Fluor 5 650 nm 665 nm Follow the instructions provided with your scanner for adjusting the photomultiplier tube PMT settings It is important to adjust the PMT setting for each channel for maximum dynamic range and channel balance
26. h removes more debris from the sample enhancing the performance of this kit e We recommend starting with high quality isolated small RNA We do not recommend using total RNA for amplification labeling and hybridization TM The following protocol has been adapted from the PureLink miRNA Isolation Kit manual and may be used to isolate miRNA from up to 10 ug of total RNA TM See the PureLink manual for protocols for isolating miRNA from cells or tissue Materials needed e Components of the PureLink miRNA Isolation Kit Invitrogen catalog no K1570 01 see page vii e Total RNA sample e 70 ethanol e 96 100 ethanol e Microcentrifuge e 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 ul of Binding Buffer L3 supplied with the PureLink kit Mix well by vortexing or pipetting up and down Add 300 ul of 70 ethanol to the solution Mix well by vortexing Add the complete solution 600 ul to a Spin Cartridge preinserted in a Collection Tube from the PureLink kit 4 Centrifuge the Spin Cartridge at 12 000 x g for 1 minute at room temperature to collect the flow through Remove and discard the Spin Cartridge Do not discard the flow through 5 Add 700 ul of 96 100 ethanol to the flow thro
27. ial 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 Goff L A Yang M Bowers J Getts R C Padgett R W and Hart R P 2005 Rational probe 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
28. ion Kit In this step you isolate small cellular RNA molecules from biological samples using a method of choice e We recommend isolating and quantifying the small RNA in the morning and then labeling in the afternoon of the same day to avoid freeze thawing and prolonged hybridization times e Although the Poly A Tailing Procedure on page 11 includes instructions for adding NCode Multi Species miRNA Microarray Controls to the tailing reaction you can add 1 ul of diluted controls to each sample prior to isolation of small RNA to verify your isolation procedure See the documentation provided with the controls for details When working with 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 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 co
29. lide container just prior to use and close immediately to store unused slides TM e The NCode Multi Species miRNA Microarray is printed on the same side of the slide as the barcode Continued on next page 15 Tagged miRNA Hybridization continued Before Starting Tagged miRNA Hybridization Procedure 16 TM The following items are supplied in the NCode miRNA Labeling Module e 2X Hybridization Solution e DEPC treated water The following items are supplied by the user TM e NCode Multi Species miRNA Microarray ordering information on page vii or NCode miRNA Microarray Probe Sets ordering information on page vii printed on epoxy coated glass slides e g Corning Epoxide Coated Slides with barcode catalog 40041 and without barcode catalog 40044 e Hybridization chamber e g Corning Hybridization Chamber catalog 2551 or 40080 e Lint free laboratory wipes TM e Raised edge coverslips for the NCode Microarray we recommend 25 x 60 LifterSlips Erie Scientific catalog 25x601 2 4789 e Incubator or water bath at 70 C e Incubator or water bath at 75 80 C e Incubator at 52 C e Vortex mixer e Microcentrifuge Use the following procedure to hybridize the purified tagged miRNA from Purification Procedure Step 10 page 14 to the NCode Multi Species miRNA Microarray This protocol may also be used with any epoxy coated glass slide printed with miRNA probes 1 Thaw
30. lide might break Proceed to Hybridization of Alexa Fluor Capture Reagents next page 19 Hybridization of Alexa Flour Capture Reagents Introduction Following the array wash hybridize the Alexa Fluor Capture Reagents to the tagged miRNA using the procedure in this section The following procedure has been designed for use with epoxy coated glass slide microarrays If you are using a different type of array use hybridization Note solutions and protocols appropriate for that array TM Before Starting The following items are supplied in the NCode miRNA Labeling Module e Alexa Fluor 3 Capture Reagent and Alexa Fluor 5 Capture Reagent e 2X Hybridization Solution e DEPC treated water The following items are supplied by the user e Hybridization chamber e g Corning Hybridization Chamber catalog 2551 or 40080 e Lint free laboratory wipes e Raised edge coverslips e g LifterSlips Erie Scientific catalog 25x601 2 4789 e Incubator or water bath at 50 C e Incubator or water bath at 70 C e Incubator or water bath at 75 80 C e Incubator at 62 C e Vortex mixer e Microcentrifuge Alexa Fluor 3 and Alexa Fluor 3 and Alexa Fluor 5 Capture Reagents contain branched DNA Alexa Fluor 5 polymers dendrimers each with a core that consists of a matrix of double Capture Reagents stranded DNA and an outer surface comprised of hundreds of singled stranded arms The surface arms carry
31. m the tailed miRNAs for use in real time quantitative PCR qPCR SYBR Green or SYBR GreenER reagents may be purchased separately Continued on next page 3 Overview continued NCode System Workflow Diagram miRNA Isolation PureLink miRNA Isolation Kit or alternative purification method Starting material 210 ug total RNA or equivalent cells tissue miRNA Amplification NCode miRNA Yes Amplification System miRNA Microarray Control NCode Multi Species miRNA Microarray Control V2 miRNA Labeling and Detection NCode miRNA Labeling System Pre printed Self printed Preprinted or self printed microarrays NCode Multi Species miRNA Microarray V2 NCode Multi Species miRNA Microarray Probe Set V2 Verification of results further analysis qRT PCR NCode SYBR Green miRNA qRT PCR Kit or NCode SYBR GreenER miRNA qRT PCR Kit or NCode miRNA First Strand cDNA Synthesis Kit Continued on next page Overview continued Materials Supplied The following materials are supplied by the user by the User 10 ug of total RNA or eguivalent cells or tissue or amplified miRNA from the NCode miRNA Amplification System Recommended PureLink miRNA Isolation Kit see page vii Recommended Quant iT Ribogreen RNA Assay Kit or the RediPlate 96
32. n Cartridge at maximum speed for 1 minute at room temperature 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 Quantifying Small RNA Introduction Quantifying the Amount of Small RNA Expected Yields with PureLink Kit 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 You must determine the quantity of isolated small RNA prior to poly A tailing This quantity is used to determine the amount of ATP to use in the poly A tailing procedure see next page 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 vii Each kit provides highly accurate fluorescent quantification of minute quantities of RNA in the range of 1 1 000 ng ml A 1 20 dilution of 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
33. ng overnight hybridization of the tagged miRNA perform the array wash procedure in this section prior to hybridization of the Alexa Fluor Capture Reagents The following items are supplied by the user e Wash Solution 1 2X SSC 0 2 SDS may be prepared from UltraPure 20X SSC and UltraPure 10 SDS Solution available from Invitrogen see page vii e Wash Solution 2 2X SSC e Wash Solution 3 0 2X SSC e Clean slide rack e 3clean wash containers capable of completely submerging an array slide in a slide rack e g 400 ml e An additional wash container or squirt bottle for washing off the coverslip e Incubator or water bath at 50 C e Vortex mixer e Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack or with a slide holder Prepare appropriate volumes of Wash Solution 1 2X SSC 0 2 SDS Wash Solution 2 2X SSC and Wash Solution 3 0 2X SSC prior to beginning the wash procedure Prepare enough of each solution to fully submerge the slide in a slide rack in a wash container filled with the solution The table below provides volumes for preparing 2 liters of each solution from UltraPure 20X SSC UltraPure 10 SDS and dH50 Wash Solution 1 Wash Solution 2 Wash Solution 3 UltraPure 20X SSC 200 ml 200 ml 20 ml UltraPure 10 SDS 40 ml dH 0 to 2 liters to 2 liters to 2 liters The following procedure has been designed for use with epoxy coated glass slide microa
34. ntamination from surfaces For further information on controlling RNase contamination see Ausubel et al 1994 Sambrook et al 1989 We recommend using the PureLink miRNA Isolation Kit Invitrogen catalog no K1570 01 see page vii to isolate small cellular RNA molecules from biological samples The NCode miRNA Labeling System was developed and TM optimized using enriched miRNA from the PureLink kit TM 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 We recommend isolating small RNA from 10 ug of total RNA or equivalent cells or tissue Continued on next page Isolating Small RNA continued Amount of Isolated Small RNA Reguired Important Isolating Small RNA Using the PureLink miRNA Isolation Kit Typically 10 ug of total RNA or equivalent cells or tissue yields 200 600 ng of small RNA molecules depending on the sample We recommend using this amount for each sample hybridized to the array A dual color hybridization would require two samples of 200 600 ng each For smaller amounts of starting material use the NCode miRNA Amplification System see page vii e When using the PureLink miRNA Isolation Kit to isolate small RNA use TM the protocol below which has been adapted from the standard PureLink protocol Note that the following protocol uses 100 ethanol whic
35. om the hybridization chamber Step 8 page 17 and holding the slide at a downward angle gently sguirt the wash solution onto the surface until the coverslip falls off Quickly transfer the slide to the slide rack in prewarmed Wash Solution 1 from Step 2 Make sure the slide is completely submerged in the solution Do not expose the slide to air for more than a few seconds to avoid air drying Wash the array in Wash Solution 1 for 10 15 minutes at room temperature with gentle agitation 150 200 rpm Fill a clean wash container with Wash Solution 2 at room temperature Transfer the slide rack with the slide to this wash container and wash with gentle agitation at room temperature for 10 15 minutes Do not expose the slide to air for more than a few seconds and make sure that the slide is completely submerged in Wash Solution 2 Fill a clean wash container with Wash Solution 3 at room temperature Transfer the slide rack with the slide to this wash container and wash with gentle agitation at room temperature for 10 15 minutes Do not expose the slide to air for more than a few seconds and make sure that the slide is completely submerged in Wash Solution 3 Important Perform the next step quickly To avoid high background on the array do not allow the array surface to air dry prior to centrifugation Prepare a centrifuge with a microtiter plate rotor adapter that will accept the slide rack containing the array slide Balance the opposin
36. or your product 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 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 consequent
37. rrays If you have hybridized the tagged miRNA to a different type of microarray use solutions and protocols appropriate for that array Always wear powder free latex gloves when handling arrays Avoid contact with the printed array surface The array surface should remain as lint free and dust free as possible Continued on next page Array Wash continued Array Wash Procedure Follow the procedure below to wash the array slide hybridized with tagged miRNA from Tagged miRNA Hybridization Procedure Step 8 page 17 1 Prewarm the prepared volume of Wash Solution 1 see previous page to 50 C 2 Placea slide rack in a wash container and fill the container with prewarmed Wash Solution 1 until the rack is completely submerged in the wash solution 3 To wash the coverslip from the array slide use one of two methods e Fill another wash container with prewarmed Wash Solution 1 Remove the array slide from the hybridization chamber Step 8 page 17 and holding the slide at the barcode end submerge it in Wash Solution 1 Gently move it back and forth in solution until the coverslip falls off e Filla squirt bottle with prewarmed Wash Solution 1 Remove the array slide from the hybridization chamber Step 8 page 17 and holding the slide ata downward angle gently squirt the wash solution onto the surface until the coverslip falls off 4 Quickly transfer the slide to the slide rack in prewarmed Wash Solution 1 from Step 2 Mak
38. sidual Wash Buffer Remove the collection tube and discard Place the Spin Cartridge onto a new RNase free 1 5 ml Collection Tube supplied in the kit Add 20 ul of DEPC treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute Centrifuge at maximum speed for 2 minutes to collect the purified tagged miRNA The eluate contains your purified tagged miRNA Proceed directly to Tagged miRNA Hybridization next page Tagged miRNA Hybridization Introduction NCode miRNA Microarrays and Probe Sets Note on Hybridization Temperature Note Important In this step you hybridize the purified tagged miRNA to an epoxy coated glass slide printed with miRNA probes in the antisense orientation The hybridization reagents in this kit have been developed and optimized for use with epoxy coated glass slides printed with NCode miRNA Microarray Probe Sets Probe sets are available separately or as part of the NCode Multi Species miRNA Microarray for ordering information see page vii e The NCode Multi Species miRNA Microarray is a Corning Epoxide Coated Glass Slide printed with approximately 900 unique probe sequences for miRNAs from six species human mouse rat D melanogaster C elegans and Zebrafish Probes target all the miRNAs in Sanger mirBase 7 0 http microrna sanger ac uk as well as 144 predicted human miRNAs that have been designed using comparative regulatory motif an
39. t page 21 Hybridization of Alexa Flour Capture Reagents continued Hybridization of Alexa Fluor Capture Reagents Procedure continued 22 Procedure continued from previous page Place the washed array slide from Array Wash Procedure Step 8 page 19 face up in an open hybridization chamber Important When performing the following steps to the extent possible shield the array from direct light to avoid photobleaching of the capture reagents 6 Gently vortex and briefly centrifuge the Hybridization Mix Then either position a LifterSlip on the array and pipet the hybridization mix under the slip or pipet the hybridization mix onto the array and then apply the coverslip as previously described in Step 6 Tagged miRNA Hybridization page 17 Once again be careful to avoid the formation of bubbles under the coverslip 7 Add the appropriate amount of DEPC treated water or 2X Hybridization Solution to the hybridization chamber to maintain humidity and seal the chamber Maintaining controlled humidity during hybridization is crucial to prevent the slide from drying out TM 8 Place the hybridization chamber in an incubator at 62 C for NCode arrays Incubate in the dark for 4 hours During incubation you may prepare Wash Solution 1 and begin prewarming it to 60 C see Step 1 Final Array Wash Procedure page 24 Proceed directly to Final Array Wash next page Final Array Wash Introduction Before Starting
40. the length of the slide Then position your pipette tip along an open short edge of the LifterSlip and slowly and carefully pipet the volume of prepared Hybridization Mix from Step 2 under the LifterSlip until the array surface underneath is completely covered with the mix When pipetting be careful not to form bubbles under the slip If bubbles appear you may try to remove them by gently tapping the LifterSlip with a pipette tip e Alternatively for either LifterSlips or non raised edge coverslips pipet the volume of prepared Hybridization Mix down the center of the array and then carefully apply the coverslip Be careful not to form bubbles under the slip If bubbles appear you may try to remove them by gently tapping the coverslip with a pipette tip 7 Add the appropriate amount of DEPC treated water or 2X Hybridization Solution to the hybridization chamber to maintain humidity and seal the chamber Maintaining controlled humidity during hybridization is crucial for successful microarray experiments to prevent the slide from drying out 8 Place the hybridization chamber in an incubator at 52 C and incubate overnight 16 20 hours During incubation you may prepare Wash Solution 1 and begin prewarming it to 50 C see Step 1 Array Wash Procedure page 19 The next day proceed to Array Wash next page 17 Array Wash Introduction Before Starting Prepare Wash Solutions Note Important 18 Followi
41. ual wash Transfer the slide guickly between wash background on the solutions dried on containers and centrifuge immediately after the array microarray slide final wash step Avoid exposing the slide to air between washes for more than a few seconds Dried wash solution will appear as streaks on the slide Dehydration of the This freguently appears as high background Hybridization Mix around the edges of the LifterSlip coverslip Make sure that the Hybridization Mix completely covers the array surface under the LifterSlip coverslip and that humidity is maintained during incubation Improper array Always wear powder free gloves when handling handling the array and avoid touching the slide surface Insufficient blocking of If you are printing your own arrays carefully TM the array prior to follow all array blocking procedures NCode hybridization Multi Species miRNA Microarrays come fully blocked and ready to use Poor slide quality Arrays scanned prior to hybridization should show no fluorescence Scan a slide from each printing batch prior to hybridization 27 Purchaser Notification Limited Use Label License No 280 DNA Microarray Trademarks of Other Companies 28 Manufactured under license from Genisphere Inc TM 3DNA is a trademark of Genisphere Inc Corning is a registered trademark of Dow Corning Corporation GenePix is a registered trademark of Molecular Devices Corporation SpeedVac
42. ugh and vortex to mix Transfer 700 ul of the sample from Step 5 to a new Spin Cartridge in a Collection Tube Procedure continued on next page Continued on next page 7 Isolating Small RNA continued Isolating Small Procedure continued from previous page RNA Using the 7 PureLink miRNA Isolation Kit continued 10 11 12 13 14 15 16 17 18 19 Centrifuge the Spin Cartridge at 12 000 x g for 1 minute at room temperature Transfer the remaining sample from Step 5 to the Spin Cartridge from Step 6 and centrifuge at 12 000 x g for 1 minute at room temperature Discard the flow through and re insert the Spin Cartridge into the Collection Tube Add 500 ul of Wash Buffer W5 prepared with ethanol see above to the Spin Cartridge Centrifuge 12 000 x g for 1 minute at room temperature Repeat Steps 10 11 one more time Discard the flow through and place the Spin Cartridge into a Wash Tube supplied with the kit Centrifuge the Spin Cartridge at maximum speed for 2 3 minutes at room temperature to remove any residual Wash Buffer Discard the Wash Tube Place the Spin Cartridge into a clean 1 7 ml Recovery Tube supplied with the kit Add 50 100 ul of sterile RNase free water pH gt 7 0 supplied with the kit to the center of the Spin Cartridge higher elution volumes may increase yield but will result in more dilute sample Incubate at room temperature for 1 minute Centrifuge the Spi
43. use with epoxy coated glass slide microarrays If you have hybridized the tagged miRNA to a different type of microarray use solutions and protocols appropriate for that array e During this procedure minimize exposure of the hybridized array to direct light to avoid photobleaching e Always wear powder free latex gloves when handling arrays Avoid contact with the printed array surface The array surface should remain as lint free and dust free as possible Continued on next page 23 Final Array Wash continued Final Array Wash Procedure 24 Follow the procedure below to wash the array slide from Hybridization of Alexa Fluor Capture Reagents Procedure Step 8 page 21 Be careful to minimize exposure of the hybridized array to direct light to avoid photobleaching 1 Prewarm the prepared volume of Wash Solution 1 see previous page to 60 C Place a slide rack in a wash container and fill the container with prewarmed Wash Solution 1 until the rack is completely submerged in the wash solution To wash the coverslip from the array slide use one of two methods e Fill another wash container with prewarmed Wash Solution 1 Remove the array slide from the hybridization chamber Step 8 page 17 and holding the slide at the barcode end submerge it in Wash Solution 1 Gently move it back and forth in solution until the coverslip falls off e Filla squirt bottle with prewarmed Wash Solution 1 Remove the array slide fr
44. ybridization properties Goff et al 2005 Each slide comes blocked and ready to use The NCode Multi Species miRNA Microarray Probe Set V2 includes the probe sequences provided on the NCode microarray 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 V2 is a synthetic 22 nucleotide miRNA sequence that has been designed and screened as a positive control for use with NCode system This control sequence has been tested for cross reactivity 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 quantitative RT PCR qRT PCR This kit has been optimized for the detection and quantification of miRNA from 10 ng to 2 5 ug 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 qRT PCR This kit has been optimized for the detection and quantification of miRNA from 10 ng to 2 5 ug 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 fro
45. ybridize the Alexa Fluor Capture Reagents to the TM NCode Multi Species miRNA Microarray This protocol may also be used with any epoxy coated glass slide printed with miRNA probes 1 Thaw the 2X Hybridization Solution by heating it at 70 C for 10 minutes and then vortexing to resuspend evenly If necessary repeat heating and vortexing until the solution is fully resuspended When resuspended centrifuge for 1 minute Prepare the Hybridization Mix Use Alexa Fluor Capture Reagents thawed as described above The amount of Hybridization Mix per array depends on the coverslip size Note The following table shows amounts for a dual color hybridization For single color hybridizations replace the volume of the second capture reagent with DEPC treated water Amount per Array Component 24 x 50 Coverslip 25 x 60 Coverslip Alexa Fluor 3 Capture Reagent 2 5 pl 2 5 pl Alexa Fluor 5 Capture Reagent 2 5 pl 2 5 ul DEPC treated water 15 pl 20 ul 2X Hybridization Solution 20 ul 25 ul Final Volume 40 ul 50 ul Gently vortex and briefly centrifuge the Hybridization Mix then incubate in the dark at 75 80 C for 10 minutes After incubation hold the mix at the hybridization temperature 62 C in the dark until loading the array Wearing powder free latex gloves inspect the coverslip to ensure it is clean If necessary gently wipe clean with a lint free laboratory wipe Procedure continued on next page Continued on nex
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