实验常用protocol英文版

miRNAUsingamiRNAmicroarraycanbebrokendowninto5Post-Processing(CanbedoneaheadofIsolationofSmallRNAsfromsample(CanbedoneaheadofLabelingofRNA hedayofWashingandScanning hedayafterNote:YouwillbeapplyingRNAdirectlytothearray,alwaysusegloveswhenhandlingthearrays.Arraysshouldbestoreddesiccatedatroomtemperature.Post-Processing—Afterpost-processingarrayscanbeusedforuptoseveralMetalslideRe-hydrationtrays(SigmaH6644)CentrifugewithsliderackadaptorsSuccinicanhydride1MSodiumBorateSolution,pH8.0.(adjustpHwithNaOH)Diamond-tippedglassetchingpen(VWR52865-005)StrataLinkerforUVcross-linkingPour100ml0.5xSSCintohydrationtrayandwarmonslidewarmer.Setthewarmerto37°CPre-heataheatingblockatmaxAfterprocessing,thearrayswillnotbevisible,sotheirboundariesneedtobemarked.Holdingthediamondpenperpendiculartotheslide,marktheboundariesofthearrayonbacksideoftheslide.Setslidearraysidedownonthehydrationtrayandobservespotsuntilfullhydrationisachieved(thiswilllooklikealightlayerofcondensationcoveringthearray).Donotrehydratemorethan1min.Uponreachingfullhydration,drytheslidesbyfliponeatatimeontotheheatingblockwiththearrayfaceup.Dothisinonesmoothmotion,withonehand,p hingthearrayatoneendandflipitoverasyoumoveittothehotte.Havingagoodbrightlightattherightanglewillhelpyouseetheslidedrying.Thearrayshoulddrywithin1-2seconds.Removetheslideandceintoametalsliderack.UVcrosslinkthearraysat60mJ(ifyouareusingastratalinker,pushtheenergybutton,lighting-uptheindicatorforujoulesx100,enter600andthenpressstart).Measure335mlof1-methyl-2-pyrrolidinoneintoaclean,dry500mLbeaker.Dissolve5.5gofsuccinicanhydrideinthe1-methyl-2-pyrrolidinoneusingastirbar.Notethatthestockbottleofsolidsuccinicanhydrideshouldbestoredunderdesiccationandvacuum.Donotuseifexposedtomoisture,avoidusingIMMEDIAYaftersuccinicanhydridedissolves,mixin15mlof1MsodiumboratepH8.0.Quicklypourthebufferedblockingsolutionintoaclean,dryglassslidedish.Plungetheslidesrapidlyintoblockingsolutionandvigorouslyshake,keethetopsoftheslidesunderthelevelofsolution.After30secondsofplunge-mixing,putalidontheglassbox,andletshakegentlyonarotatorfor15minutes.Transferslideracktoaglassjarfilledwithroomtempdistilledwater.Plungetheslidesupanddownafewtimesinthewater.Transfertheracktoaglassdishof95%EtOHandplungeseveraltimestorinse.MakesuretheEtOHiscrystalclear.Donotuseifitappearstohaveparticulatesorappearscloudy.Spinsliderackinabenchtopcentrifugefor1minuteat550Afterspinning,theslidesshouldbecleananddry(ifnotdryyoucantryspinningagain,ifnotcleanyoucantryre-washinghanolfollowedbyanotherspin).IsolationofSmallRNAsfromsample—itisusuallyadvisabletocheckfordegradationofyourRNAeitherafterextractionorbeforethisstep.ThiscanbedoneonageloronanAgilentBioyzer.TherearetwooptionsforsmallRNAisolation.1)flashPage,anAmbionproduct,whichisquickbutnotefficientforrunningmanysamplesinparallel,or2)acrylamidegelsizefractionation—slow,butmanysamplescanberuninparallel.PurifyRNAsof<40ntforuseonthearrayusingAmbion’sflashPAGEelectrophoresisunitaccordingtotheirprotocol:.RNAscanthenberecoveredbythe )clean-upkitorbyprecipitation )(Note:Douseaglycogencarrier). allRNAonadenaturingacrylimide10%DenaturingPolyacrylamideGelTrizolPurifiedRNASamples(50-500ug)GelLoadingBuffer(withbromophenolblueandxylenecyanol)3MSodiumAcetate15mLFalconRunthegelatconstant65mAuntilthegelreachesaboutMixthesampleRNAwithequalvolumeofgelloadingbufferandheatat70Cfor2min,andputonice.Rinseoutureawithreservoirbuffer.Runthegeluntilthereisabout1.5cmseparatingthexylenecyanolandbromophenolbluebands.Cutouttheslic weenthebandsandcutapartseparatesamples.Useasyringeplungertocrushthegel. ceina15mLfalcontubeandadd1mLof0.3Msodiumacetateforeverycminwidthoflaneusedtorunsample.RockovernightatCentrifugetubefor5minutes(2000xg),collectsupernatantandputat-Addtothegel1/5thevolumeofsodiumacetateusedforthefirsution.Rockfor1houratroomtemperature.Centrifugeasbefore,collectthesupernatantandaddtothesupernatantfromtheAdd4volumesof100%ethanoltothecollectedsupernatant.ubateat-20CCentrifugeat16,000xgatDiscardWashthepelletwith500L75%icecoldethanol.Centrifugeat16,000xgfor10minutesat4C.Discardthesupernatantandairdrythepellet.LabelingofRNA—thisisbestdonejustbeforehybridization,althoughthereisonepossiblestoppointintheprotocol,afterwhichtheRNAcanbestoredfor1-2days.LabelsmallRNAsusingAmbion’smirVabelingkitaccordingtotheir1562.pdf.Note:TheCy3andCy5fluorophoreswillphotobleach,itisbesttominimizetheirexposuretolightasmuchaspossible—thisludesthehybridizedarray.TheamountofRNAtouseforhybridizationwillvarydependingontheconcentrationofthemiRNAsthatyou’retryingtovisualize.HybridizingwiththesmallRNAsisolatedfrom20goftotalRNAmightbeagoodstartingpoint.LabeledRNAWaterbathat42CHeatBlockat100CAmbion3xhybridizationbuffer(Cat#1567mirVana™miRNABioarrayEssentialsKit)22x25mmlifterslips(Erie22x25I-M-5226)Heat3xhybbufferat65CandvortextoAdd5to10100Ldropsof5xSSCinthebottomofthehybridizationchambertopreventthearraysfromdryingout. cetheslidesinthehybridizationchamber,arraysideup.Addthelifterslipoverthearrays(thisiswherethediamondpenmarkingcomesinhandy).Aflatmetalspatulacanbehelpfultoalignonesideofthelifterslip,sothatyoucangentlydroptheothersidedown.Usethespatulatoalignthelifterslipoverthearray.Becarefulnottoscratchthearraysurface. hevolumeofelutedRNA(shouldbearound20L)andaddhalfvolumeinhybbuffer(ie.fora20lsample,add10Heatdenaturesamplesina95Cheatblockfor2minutes.Microfugeto yapplyClosehybridizationchamber,carefullytransferimmedia ytoa42Cwaterbathfor12-16hours.AmbionSaltConcentrate(Cat#1567mirVana™miRNABioarrayEssentialsAmbionDetergentConcentrate(Cat#1567mirVana™miRNABioarrayEssentialsKit)Metalsliderack4Glassslideboxeswithcovers(inwhichtheslideracksfit)CentrifugerotoradaptorsformetalslideracksPrepare4slideboxeswithwashingsolutions.Thefirsttwoarelowstringency(4mLdetergentconcentrate,20mLsaltconcentrate,and376mLH2O)andthesecondtwoarehighstringency(20mLsaltconcentrateand380mLH2O).CarefullyremovehybridizationchamberfromthewaterRemoveslidesoneatatimefromthechamber,tipofflifterslipinfirstlowstringencybathandimmediaycetheslidesinthemetalracksittinginthe2ndlowstringencybath.Washslidesinthelowstringencybath,byplungingthemupindowninthesolutionfor1minute(donothitthebottomoftheboxtoohardoryouwilldamageyourslides).Washinfirsthighstringencybathfor1Washinsecondhighstringencybathfor1Spinsliderackinabenchtopcentrifugefor3minutesat800ceslidesinasticslideboxandscanimmediaNote:MuchofthisprotocolhasonlyseenminoradaptationsfromprotocolsofMichaelMcManusandCarolineMrejen,manythanksfortheirassistance.miRNAmiRNA5RNA（杂交当天完成(SigmaH6644)1-甲基-21M，pH8.0（用NaOHpH）VWR52865-005)UVStrataLinker方案a)100ml0.5xSSC37°C薄薄的凝结物）。补水时间不要超过1分钟。1-2UV60mJ（stratalinker，ujoulesx100指示灯，输入600，然后按开始）。335ml1-甲基-2500mL5.51-甲基-2-吡咯烷酮中。请注意，固体琥珀酸酐的储备瓶应在干燥和真空下保存。如果暴15ml1MpH8.0。301595%EtOHEtOH550rpm1RNA1)flashPage，Ambion率不高，或2)丙烯酰胺凝胶尺寸分级-速度慢，但可以并行运行许多样品。AmbionAmbionflashPAGE40ntRNA，用于阵列：。然后可以通过