細(xì)胞組織RNA抽提試劑盒說明書

1 1 2 2 3 3 5 7 7 8 8 9 13 QuantitationofRNA JudgementoftheQaulityofTotalR 1背景及原理介紹DxGeneTM組織和細(xì)胞系總RNA抽提試劑盒經(jīng)過一系列優(yōu)化被設(shè)計用于從各種細(xì)胞和組織（包括富含脂肪的組織）樣品中純化出高QuantitationKit,DxG因檢測試劑盒結(jié)合使用，為準(zhǔn)確定量癌癥患者中各指標(biāo)基因包括的表達(dá)情況提供優(yōu)質(zhì)的總本試劑盒將傳統(tǒng)的酚/胍鹽抽提法和硅膠裂解試劑，能促進(jìn)細(xì)胞和組織的裂解以及抑制種污染包括酚等試劑都被洗脫，從而得到高純1123是被開發(fā)和優(yōu)化用來從新鮮組織和細(xì)胞中提提取，請選擇DxGeneTMFFPETotalRNA織。注意：心3min，用1XPBS洗滌后再加入1mlEzol裂解液。Ezol裂解液。污染。45.將上清水相轉(zhuǎn)移至另一新的無RNA酶離心6.立即吸取700μl樣品以及有可能形成的沉8.往離心柱中加入700μlWB，輕蓋注意：第一次使用前請確認(rèn)是否已經(jīng)往5通常此類問題是由于加了氯仿之后不充分的混勻以及隨后在比較高的溫度下離心引起。不充分的勻漿可能導(dǎo)致大塊組織殘留堵塞純請盡量縮短處死到取樣的時間，這樣才能保在把組織樣品投入裂解液前，請保持冷凍的6道中的總RNA條帶就會出現(xiàn)彌散和拖尾現(xiàn)酶的污染，整個過程始終需戴手套，并且經(jīng)留的RNA酶。所有的離心管和吸頭必須用量的組織樣品，不充分的勻漿以及低效的洗脫。因此，選取高質(zhì)量的組織樣品并進(jìn)行合脫，適當(dāng)?shù)难娱L洗脫時的孵育溶解時間有助通常，A260/A280比值偏低是由于在水中吸收值，或者是由于酚和其他有機藥品的污不含酚和其他有機藥品。另外，不充分的勻漿可能導(dǎo)致蛋白和核酸的共純化，從而使冊中的標(biāo)準(zhǔn)流程消化和勻漿組織樣品，另一7抽提純化得到的總RNA能應(yīng)用于多種您所需要的后續(xù)實驗，如northernblotting和能給您提供更加穩(wěn)定放心的實驗結(jié)果，除此之外，本試劑盒還能保留小片段RNA，如為了得到穩(wěn)定的基因檢測結(jié)果，本試劑盒同R以為研究者提供從抽提，定量到檢測一整套便利的服務(wù)。吉瑪?shù)募夹g(shù)人員總是樂意回答您所遇到的任何問題，無論是操作步驟還是技術(shù)上的問題，我們都將給與最大的支持。8本試劑盒所抽提的RNA基本上沒有任何有機藥品的污染，因此非常適合用紫外分光光度計進(jìn)行定量。RNA的濃度可以在一個石英或者紫外適用的比色皿中通過測量260nm處的吸收水中進(jìn)行定量分析，因此RNA必須用無RNA酶水溶解，稀釋和測量，同時為了最大限度減們推薦使用超微量的比色皿，只需要消耗常規(guī)比色皿的十分之一的RNA就能進(jìn)行測量。適用于紫外測量的比色皿您可以從幾個生物公司購得：例如BrandTechScientific用戶可以選擇其他方法，比如RiboGreen法（Invitrogen）或者NanoDrop設(shè)備（Thermo結(jié)果。但是用戶們必須于測量樣品之前在RiboGreen法中先建立一條標(biāo)準(zhǔn)曲線，或者先用RNA標(biāo)準(zhǔn)品校正Nanodrop。如果用戶采用9本試劑盒提取的RNA的大小和完整性可以經(jīng)過溴乙錠染色后，膠上會呈現(xiàn)出兩條銳利注意：由于配套的DxGeneTM熒光定量檢測響。本試劑盒將傳統(tǒng)的胍鹽/酚/氯仿法和純化柱相整合，在試劑盒所述的步驟和環(huán)境下能分值可以對RNA的純度作大概評價。通常來響，因為水不是緩沖體系，因此在水中測量的A260/A280會略低于在緩沖液中測量的結(jié)果，并且結(jié)果之間會有很大的差異，結(jié)果可能導(dǎo)致一些誤解，例如樣品是否被蛋白，酚或者其他有機試劑污染，因為這些污染都在的緩沖體系中測量，并用相同的緩沖液校準(zhǔn)和RNA濃度之間的換算關(guān)系取決于RNA在我們的測試結(jié)果表明不需要再額外添加用于配套的DxGeneTM熒光定量檢測試劑萬維網(wǎng)質(zhì)量控制聯(lián)系我們我們有嚴(yán)格的質(zhì)量控制體系，每一批產(chǎn)品都經(jīng)過MCF7和?2009-2010GenepharmaCorporation.Allrightsreserved.Forresearchuseonly.NotintendedforanyanimalorhumantherapeuticordiagnosticustotalRNAofhighqualityextractionfromcelllinesandtissuesamplesincludinprovidingtotalRNAofhighqualityandquatitationofHer2andEGFRaccuratelyincellandtissExtractionKitintegratestrsilica-gel-membranepurificationoftotalRNA.ThekitalsoprovidesEzolTMreagent,designedtofacilitatelysisofsinhibitRNase.AfterRNAbindingtothesilicamenbrane,phenolandothercontkit.Thenhighqualityof50μlormoreRNase-freewater.RNase-freeEutionBufferUserManual11EquipmentandReagentstoBeWhenworkingwithchemicals,alwayswearalabcoat,disposableglovesandprotecRNase-freewater/DEPCwaEqiupmentandtubesfortissuediMicrocentrifuges(witharotorfor2mDxGeneTMHer2DuplexTestKitaGenePharma.YoucancontactCat.No.ItemExtractionKitisdevelopedandoptotalRNAisolationsamples,pleaseselepieceofdeep-frozentissueinachilledmortar,andweightbybalance.Transfertissueialreadycontains1mlofEzoHomogenizetissuewithanelectronvisibletissue,andthenmoveNote:Tissuesampleshouldbestoredinliquidnitrogenorindeepfreezer(-80oC).ThisprotocolissuitableforisolationofRNAfromlessthan50mgoftissue;otherwise,moreEzolreagentisneededtodigesttissuesample.culturedishorffNote:PBSbufferisnotprovidedwithkit,andisavailableseparaNote:Forsuspensioncells,spindowncellsfirstat2,000xgfor3min,washcellstwicewith1XPBS,andthenadd1mlofEzolreagent.Note:ThisprotocolissuitableforisolationofRNAfrom≤1x107cells,orfroma≤10-cmdiameterdish;otherwise,morereagentisneededtohomogeniz3.IncubatemixtureatroomtNote:AvoidvertexingasthismayincreasetheDNAcontaminationtotheRNAsa4.PlacethetubeatroomtemperatureNote:Centrifugationseparatessolutionintothreephases:upperaqueouswithRNA,middlelayerwithprotein/DNA,bottomorganicwithDNA.The4oCisessentialforphaseseparation.volumeof70%ethanol.MixsNote:DiscardthemiddlelayerandbottomorganicphaseifnoneedforisolationofDNA.immediately,includinganyprecipthatmayhaveformed,intoamini-sClosetubegently,atemperature.Discardtheflow-throsample,anddiscardtheflow-through.Note:Ensurethatthreevolumeof100%ethanolisaddedtoWBbeforeusingforthefirsttime.buffer.Afterlastwash,centrifugesilica-gelmembrane.Note:Itisimportanttocompletelyremovewashingsolution.AnycarryovermayaffectdirectlyontotheRNeasysilic Kitiscarefullyassembledandcontainshighqualityofreagents.ThistroubleshootingistoprovideageneralguideforsolvingtheproblemswhichareoftenrelatedtotisManyotherproblemsmayoccurduringexproblemwithourtechniqueservice.Phasesdon’tseparatecomInsufficienthomogenadditionofchloroform,orbycentrifugationataoftheprotocol,youneedtoshakethetubefhigherthan8oC.AhighertemperaturewilldisruptphaseseparatioInsufficienthomogeInsufficienthomogenattissuepiecethatmayclogtheincreasetheg-forceandtimeofcentrifugationifnecessary.Thestartingmaterialcann100mgtissue.Pleasereducetheamountofstartingmaterialifnecessary.DegradationofRNAtrogen.ItshouldbebetterfsomecommercialRNaseinhipossibleonceshortenthetimeofcollectionaspossibleasyoucantoyieldhigherqualityoftotalRNA. eptissuefrozenbeforricebathimmediatelPleasecentrifugesamplesDegradationaftereExogenousRNasecontamiIfloadingRNAismorethan5μg,thebandwillto800ngtotalRNAforeleDisposableglovesandduringthewholeprocesstopreventanyRNasecontamination.Themortarandmullershouldbedthenautoclavethembeforeuse.RNAisdegradedbyribonucleases(RNases)whichareverystableanthematerialsandreagentsprovidedbykitarefreeofRNworkingenvironment,suchasonhands,laboratorybench,andperceivablethatRNAsamplescouldbecontaminatedbyRNasesduringorafterisprocedure.Wesuggestyoutakefollowingprecautionstoavoidiinhibitor-containingsolution;3)ARNAsamples;4)Collecttissuesamsampleat-70oCinaliquottoreducefreeze/thawcyclgelrunning;8)AlwaysdiluteRNAsampleswithRNase/DNase-freewaterprovideLowYieldindicatefailureofextracreducetheyieldofRNA,suchaspoftissuesamples,insufficienthomogenization,qualitytissueandhandleitaCompletehomogenizationinEzolreagentis elution,andanextendedincubatLowA260/A280ValueUsually,alowA260/A280valueiscausedbymeasurementofabsorbanceinwater,orbycontaminationofproteins,phenol,orotherorganicchemicals.Undernormalconditions,homogenizationmaycauseco-purificationofrecommendfollowingthedescribedprocedalsorecommendmeasuringtheratioofIV.DownstreamApplicationIsolatedtotalRNAcanbeappliedfkindsofexperimentssuchasreal-timePCR,northernblottingorgenechipthatyouneed.Thehighpurityandquantprovideyoumoreconsistentresult.Bethatyoucanalsodetectforreforresearchertodealwithsampleextraction,quatitationtothedetection.Theeithertheinformationortheprotmanualandotherrelatedproducts.PleaseseetheappendixformorecontactinfThereisnoneedtoaddanyothesuchasRNaseinhibitor-70oC.Storageat-7long-termusage.Underthiscondition,noobviousdegradationofRNAisdetebyfrequentfreeze/taliquotedbeforestorage.QuantitationofRNARNAsamplespreparedbythiskitarebasicallyfreeofcontaandtherefore,arewellsuitabofRNAcanbedeterminedbymeasuringtheabsorbanceat260nm(A260)usingquartzorUV-compatiblecuvettes.Anabsorbanceof1unitat260nmiThisrelationshipisvalidonlyinwater,RNase/DNase-freewater.Tominimizevariation,readingsofsampleabsorbausingultra-microcuvettes,whichconsumeasurementinregularcBiotechnology().Beforemeasuringsamples,wealsorspectrophotometerwithRNAstandardsolution,whichisavailablesNote:AnexampleofdeterminationofRNAconcentrationusingregularcuvette:TotalvolumeofRNAsample,A260Reading,0.20(measuredina1-mlcuvette)And,TotalyieldofRNAsample=800x0.05=40μgDeterminationofRNAconcentrationforthesamesampleusingultra-microcuvette:TotalvolumeofRNAsample,A260Reading,0.20(measuredinanultra-microcuvette)So,ConcentrationofRNAsample=40xA260xdilutionfactorAnd,TotalyieldofRNAsample=800x0.05=40μgneedtoprepareaRNAstandardcurveinRiboGreenassaRNAstandardbeforemeasuringsamples.CustomemanualifothermethodsareusedforquantitationofRNAsamples.JudgementoftheQaulityofTotalTheintegrityorthesizeoftotalRNAagarosegelelectrophoresis,bandsongelfollowingethidiumbromiderRNA.IfasampleshowsgoodintegrityofmoreaccurateanalysisofRNAintegrity.Note:GiventhatGeneMedDxTMreal-timePCRkitsaredesignedtoamplifythemRNAfragmentsofshorterthan100nucleotides,acomprRNAintegrityisexpectedtohavemarginalimpactonthereal-timePCRassay.guanidine/phenol/chloroformextractionmethodandcolumnpurificationproc