Analysis of RNAProtein Complexes by RNA Coimmunoprecipitation and RTPCR Analysis from Caenorhabditis elegans

INTRODUCTION

RNAcoimmunoprecipitation(co-IP)experimentsareanextensionofproteinco-IPexperimentsinwhichinvivoRNA-proteincomplexesareinvestigated.ThisprotocoldescribeshowtoperformRNAco-IPsfromC.eleganswhole-wormextracts.Inprinciple,aprotein-specificantibodyisusedtopurifytheproteinofchoiceanditsassociatedcomplexmembersfromwormextract.ThismayalsoincludeRNAmoleculesassociatedwithotherproteincomponents.ToidentifyaspecificmRNAmolecule,allRNAmoleculesarefirstseparatedfromtheproteincomponentsafterimmunopurification.ThemRNAsarethenconvertedintoCDNAbyreversetranscription.CandidatemRNAsaredetectedbysensitivegene-specificamplificationviapolymerasechainreaction(PCR)inasemiquantitativemanner.SinceRNAmoleculesareverypronetodegradation,itiscrucialtoavoidanykindofcontaminationwithRNaseactivityinthisexperiment.

RELATEDINFORMATION

Thisprotocolwasusedtoshowthatgld-1mRNAispartoftheGLD-3/GLS-1/GLD-4complexinvivo(Schmidetal.2009).Theproteinco-IPprocedureuponwhichthisprotocolisbasedisdescribedinAnalysisofInVivoProteinComplexesbyCoimmunoprecipitationfromCaenorhaBDitiselegans(JedamzikandEckmann2009).

MATERIALS

Reagents

ToprepareDEPC(diethylpyrocarbonate)-treatedsolutions,add0.1%DEPC(e.g.,SERVA18835.01)tothesolutioninascrew-capbottle.Shakethebottleveryhardimmediatelyandthenevery10minfor1h,orstirthesolutionwithastirbarfor1hifdetergentsareincluded.Autoclavethesolutions.Solutionsthatcannotbeautoclaved(e.g.,HEPES)shouldbeputintoa50°CwaterbathovernightwiththelidleftloosetoallowexcessDEPCtoevaporate.

AMVreversetranscriptase(10U/µL)and5XAMVbuffer(e.g.,Promega)

Antibodies

Specificantibodytoproteinofinterest

Nonspecificantibodyasanegativecontrol(preimmuneserumorpurifiedIgG,e.g.,ChromPureRabbitIgG,wholemolecule,JacksonImmunoResearch011-000-003)

BSA(bovineserumalbumin)

BufferB70(RNase-free)

Chloroform

Chloroform:isoamylalcohol(IAA)(24:1)

DNaseI(RNase-free)and10XDNaseIbuffer(e.g.,Roche)

dNTPs,10mM(mixfromindividualdNTPs;e.g.,FermentasR0181)

Ethanol(75%[ice-cold]and99.8%)

Gene-specificprimersfornestedPCR(seeStep50)

Glycogen(20mg/mL;e.g.,fromoyster,SERVA39766.02)

H₂O,DEPC-treated

Heparin(100mg/mLstock)(HeparinNa-salt;e.g.,SERVA24590.01)

HEPES(50mM,pH7.5),DEPC-treated

M9saltsolution

NaOAc(sodiumacetate;3M,pH5.2),DEPC-treated

Nematodegrowthmedia(NGM)plates,6-cm,containingstarvedworms

Nematodegrowthmedia(NGM)plates,10-cmand14.5-cm

OligodTprimer(12.5µM)(5""-TTTTTTTTTTTTTTTV-3"")

OP50solution

Phenol:chloroform:isoamylalcohol(PCI)(25:24:1)

ProteinA-agarose(e.g.,Roche)

Ifyourspecificantibodydoesnotcomefromrabbit,ProteinG-agaroseoranothertypemightbepreferred.Topelletagarosebeads,nevercentrifugehigherthan400g.

Reagentsforagarosegelelectrophoresis

ReagentsforBradfordassay

RNasin(40U/µL;e.g.,PromegaRNasinPlus)

Taqpolymeraseand10XPCRbuffer(e.g.TaqDNAPolymerasefromNEBM0267S)

Trizol(e.g.,TRIzol,Invitrogen15596-026)

YeasttotalRNA(20mg/mLstock;Roche10109223001)

Equipment

Adaptersfor15-mLcentrifugetubes

Centrifugetubes(15-mLglass;e.g.,Kimble45500)

EquipmentforagarosegelanalysisofDNA

Falcontubes(15-mLand50-mL,RNase-free)

Filter(0.45µm)

Gloves(protective;canbethickwintergloves)

High-speedcentrifuge(e.g.,BeckmannCoultercentrifugeAvantiJ-20orJ-25)

Iceandicewater

Laminarflowhood

Liquidnitrogen

Liquid-nitrogencontainers(2)

Magneticstirrer

Microcentrifugetubes(1.5-mL,RNase-free)

MicroPipetteandtips(including1-mLtips)

Mortarandpestle(prechilled)

PCRtubes(0.2-mLor0.5-mL,RNase-free)

Pipette(glass,drawnout)

Rotorforcentrifuging15-mLglasstubes(e.g.,JA25.50)

Sieve(metal)

Spatula(metal)

Spoon(metal)

Stirbar

Syringe(10-mL)

Tabletopmicrocentrifuge

Tabletopcentrifugefor15-mLFalcontubes(e.g.,Eppendorfcentrifuge5702)

Testtuberotationwheelfor1.5-mLtubes

Thermocycler(e.g.,MJResearchDNAEngine)

Thermoshakerpresetto65°C(e.g.,EppendorfThermomixer)

Waterbathspresetto37°Cand96°C

METHOD

Atallstepsoftheprocedure,itisimportanttoworkRNase-free.Useglovesatalltimes,treatsolutionswithDEPC,bakeglassequipment,usefilteredtipsandRNase-freeplasticware,andflamemetalequipment.Forallchemicals,haveabox/flaskthatisonlyusedforRNAwork.Donotsharesolutionswithanyone.

Large-ScaleGrowthofC.elegans

Thissection(Steps1-11)describestheculturingofalargenumberofmixed-stagewormsunderphysiologicalconditions.Toavoidanaccumulationofinhibitorypheromonesandthusadelayinwormdevelopmentoraninductionofstarvation,atwo-stepplate-basedamplificationprotocolisused.Wormsarefrozeninliquidnitrogenintheformofpearlstofacilitateusageofsmallerportions(insteadoftheentirewormculture)forextractpreparation(Step12).

1.Take1210-cmNGMplatesand3014.5-cmNGMplates,andseedeachofthemwith1mLor2mLofOP50solution,respectively.Drytheplatesunderalaminarflowhoodandstoreovernightatroomtemperature.Keepplatesat4°Cforlong-termstorage.

2.Preparethewormsfromstarved-wormplatesasfollows:

i.Takethree6-cmstarved-wormplatesandwashthewormsoffthreetimeswith1mLofM9saltsolutioninto1.5-mLtubes.

ii.Pelletthewormsbycentrifugationfor1minat400g.

iii.Putthewormsonicefor2mintosettlethem.Removethesupernatant.

iv.Washthewormsthreetimeswith1mLofM9saltsolution.

Whole-WormExtractPreparation

Thissection(Steps12-15)describesanefficientandeconomicalprotocolfortheproductionofcytoplasmicextractfromfrozenwormculturesunderconditionsthatpreservetheintegrityofcellularproteinsandRNA.Performtheentireprocedureat4°Cunlessotherwisestated.

12.Useanequivalentof2mLofsettledwormsforanIPexperimentwithtwoindividualsamples,ormoredependingonthenumberofIPsamplesintheexperiment.Grindthewormstoafinepowderinliquidnitrogenusingamortarandapestlethathavebeenprecooledwithliquidnitrogen.Maintainthewormhomogenateasacoldpasteduringtheentiregrindingprocedurebyaddingfreshliquidnitrogentothemortaronceithasevaporated.Grindthewormsseveraltimestoproduceafinepowder.Becarefulwhentouchingthecooledmortar.Itisnecessarytowearprotectiveglovestopreventcoldburns.

13.Transferthepowderinto15-mLglasstubeswithaprecooledspatulaandadd2mLofbufferB70(supplementedwithfreshproteaseinhibitors).Thepowderproducedfromanequivalentof2mLsettledwormsfillsapproximatelyhalfthevolumeofa15-mLglasstube.

14.Centrifugeasfollows:

i.Centrifugethetubesinahigh-speedcentrifugeat37,000gfor30minat4°C.

ii.Transferthesupernatanttoprecooled1.5-mLtubes.

iii.Centrifugeagaininatabletopcentrifugeat20,000gfor10minat4°C.

RNACo-IP

Thissection(Steps16-27)describeshowtoimmunopurifyRNA-proteincomplexesfromwormextractintwosteps.First,theextractisincubatedwiththeantibodiestoallowbindingoftheantibodytoitstargetprotein.Second,theantibodywiththeassociatedprotein-RNAcomplexisaffinitypurifiedusingProteinA-agarose.PerformSteps16-27at4°Cunlessotherwisestated.

16.ForeachIPexperiment(consistingofonespecificandonenonspecificantibodysample),place60µLofbedvolumeofProteinA-agaroseintoRNase-free1.5-mLtubes.

17.WashthebeadsthreetimeswithbufferB70asfollows:

i.Pelletthebeadsinatabletopcentrifugeat400gfor30sec.

ii.Removethesupernatantandaddwashbuffertothebeads.

iii.Mixbycarefullyinvertingthetubeatleastthreetimes.Avoidshakingthetubetopreventfoamproductionandfragmentingthebeads.

PurificationofTotalRNA

Steps28-38describehowtoisolateandpurifytotalRNAfromtheinputandIPsamples.Performtheentireprocedureatroomtemperatureunlessotherwisestated.

28.Addone-fifthvolumechloroform(80µLfortheinputsamplesor40µLfortheIPsamples)andshakethetubevigorouslybyhandfor15sec.

29.Letthetubesitfor3min,thencentrifugeat12,000gfor15minat4°C.

30.Transfer~60%ofthestartingvolumefromtheupperphasetoanewtube(240µLforinputand120µLforIPsamples).Avoidthewhiteinterphase!Bringthevolumeupto400µLwithDEPC-treatedH₂O.

31.Add30µLofDEPC-treatedHEPES(50mM,pH7.5),mixwell,andcentrifuge.Add400µLofPCIandmixwellbyshaking.

32.Centrifugeat13,200gfor5min.

33.Transfertheupperphase(~400µL)toanewtubewhileavoidingtheinterphase,add350µLofchloroform:IAA,mixwell,andcentrifugeat13,200gfor5min.

34.Transfertheupperphase(~380µL)toanewtubewhileavoidingtheinterphase,andprecipitatetheRNAbyadding1.5µLofglycogen(20mg/mL),1/10volumeofDEPC-treatedNaOAc(3M,pH5.2),and2.5volumesofethanol.Mixthetubewellandincubateat–20°Cforatleast30min.Atthispoint,theprocedurecanbeinterruptedandcontinuedthenextdayorlater.

35.PellettheRNAbycentrifugationat16,000gfor15minat4°C.

36.Removethesupernatantwithadrawn-outglasspipetteandwashthepelletwith1mLofice-cold75%ethanol.

37.Centrifugethetubeat16,000gfor5minat4°Candremovetheentiresupernatantwithadrawn-outglasspipette.

38.Air-drythepelletuntilitstartstoturnfromwhitetoclear,thendissolveitin20µLofDEPC-treatedH₂Oatroomtemperature.KeeptheRNAoniceandimmediatelyproceedtoStep39.

DNaseITreatmentoftheRNA

ToremovegenomicDNAcontamination,theRNAistreatedwithDNaseI,whichisdescribedinSteps39-40.PerIPexperiment,taketwoinputsamplesandtreatonewithandonewithoutDNaseItohaveacontrolfortheDNaseItreatment.

39.Assemblethefollowingreagentsin1.5-mLtubesoniceasindicated.

40.Mixthetubesbygentlytappingthebottoms,centrifugethecontentsat5000gfor10sec,andincubatefor30minat37°Cinawaterbath.ImmediatelyproceedtoStep41. RepurificationofRNA PriortocDNAproduction(Steps46-49),theRNAmustbepurifiedasecondtime(describedinSteps41-45)topreventsubsequentcDNAdegradationbyDNaseIorinefficientreversetranscription. 41.TotheDNaseI-treatedRNA,add360µLofDEPC-treatedH2O,30µLofDEPC-treatedHEPES(50mM,pH7.5),and350µLofPCI.Mixwellbyshaking. 42.Centrifugeat13,200gfor5minatroomtemperature. 43.Transfertheupperphasetoanewtubewhileavoidingtheinterphase(~400µL),add350µLofchloroform,andmixwell.Centrifugeat13,200gfor5min. 44.RepeatStep43. 45.PrecipitateandwashtheRNAasinSteps34to38.DissolveeachRNApelletin26µLofDEPC-treatedH2O.KeepthedissolvedRNAoniceandproceedimmediatelytoStep46. ReverseTranscriptionofmRNAsintocDNA ThemRNAwillbereversetranscribedusinganoligo(dT)primertominimizeribosomalRNAcontamination(Steps46-49).EachRNAsampleisdividedintoanRT+sampleandanRT–sample(negativecontrol). 46.HeattheRNAfornolongerthan3minat96°Candimmediatelyplaceitinicewaterforatleast5min. 47.Centrifugethesample,divideitintoanRT+sampleandanRT–sample,andassemblethefollowingreactionsinPCRtubeskeptonice:Addcomponentsintheorderindicated. DNaseI+ DNaseI- 10XDNaseIbuffer 4µL 4µL RNA 20µL 20µL DEPC-treatedH2O 15µL 16µL DNaseI 1µL -- 48.Mixthereactionsbygentletappingandincubatefor60minat55°C.Inactivatethereactionsfor20minat65°Cinathermocycler. 49.CentrifugethereactionsandproceedtoStep50orstorethecDNAat–20°C. Gene-SpecificPCR Duringthefollowingsteps(Steps50-54),thepresenceofaparticularmRNAintheIPandinputsamplesisdetectedinaPCRreactionwithgene-specificprimers.DuetothelowamountofmRNAinthesamples,itmaybenecessarytoperformatwo-stepPCRamplificationforhighsensitivityandspecificity.TostayinthelinearrangeofeachPCRreaction,asfewcyclesaspossIBLeareadvised. 50.Performagene-specific,nestedPCRtodetectthepresenceofaparticularmRNA(nowcDNA)inthesamples.Foroptimalresults,designtheprimerssothattheprimaryPCRproductwiththeouterprimerpairisnotlargerthan500bp(spliced!)andthesecondaryPCRproductwiththeinnerprimerpairisintherangeof~300bp(spliced!).Trytodesignprimerswithanannealingtemperatureof58°CandaGCcontentof50%. 51.Fortheinputsample,takeonesamplewith0.5µLofcDNAandonesamplewith1.5µLofcDNAastemplatetodeterminewhetherthePCRreactionisstillinthelinearrange.Bringthevolumeupto2µLwithH2O.FortheIPsamples,take2µLofcDNAasatemplateforthePCRreaction.SetupthefollowingprimaryPCRreactioninatotalvolumeof20µL: RT+ RT– 5XAMVbuffer 4µL 4µL OligodTprimer(12.5µM) 1µL 1µL dNTPs(10mM) 1µL 1µL DEPC-treatedH2O -- 1µL RNA 13µL 13µL AMVReverseTranscriptase 1µL -- Template(cDNAfromStep49) 2µL Primers(both5µMinonemix) 1µL dNTPs(10mM) 0.5µL 10XPCRbuffer 2µL Taqpolymerase(5U/µL) 0.5µL H2O 14µL 52.RunthefollowingPCRprogram: Onecycle 95°Cfor2min 10-20cycles(optimizeforeachprimerpair) 95°Cfor50sec 58°Cfor50sec 72°Cfor45sec Onecycle 72°Cfor7min Hold 4°C 53.SetupandrunthesecondaryPCRinthesamewayastheprimaryPCR(Steps51and52),butuse0.5µLofprimaryPCRreactionasatemplateforthePCR. 54.AnalyzethecompletevolumeofthesecondaryPCRreactiononastandardagarosegel.SeeTroubleshooting. TROUBLESHOOTING Problem:ThereisnosignalintheIPsamplesinthegene-specificPCR. [Step54] Solution:TheIPsamplesusuallycontainverylittleRNA(often<300ngintotal).Also,considerthefollowing: 1.RNAishighlysensitivetoanykindofdegradation.Totestwhetherdegradationisoccurringduringtheexperiment,monitortheRNAqualityofaninputsampleduringtheprocedure.AnalyzepurifiedRNAofanequivalentof1µLofextractonadenaturingagarosegel,orload1µLofpurifiedRNAonanAgilentBioanalyzerchip(e.g.,afterSteps15,18,38,and45).Anykindofdegradationshouldbeavoided(see"ThereissubstantialRNAdegradation"below). 2.TherecouldbelowenrichmentofcoimmunoprecipitatedRNAsasaconsequenceofpoorantibodyaffinity.Ifthismightbetheproblem,useahigherantibodyconcentrationortestdifferentantibodiesthatrecognizetheproteininquestion. Problem:ThereissubstantialRNAdegradation. Solution:Considerthefollowing: 1.TopreventRNAdegradationduringtheprocedure,itisimportanttoworkRNase-freeandfast.DonotinterrupttheprocedureunlesstheRNAhasbeenprecipitated(Steps34and45)orpurified. 2.KeeptheRNAoniceandavoidmultiplefreezing/thawingsteps. 3.ConsidertheadditionofthenonspecificRNaseinhibitorribonucleoside-vanadylcomplex(RVC;200mM;NEBS1402S)totheextract.RVCisanRNAanalogandgenerallyblocksRNases,whichimprovesoverallRNAstABIlity.However,EDTAmustbeexcludedinallbuffersthatcontainRVCbecauseEDTAdisassemblestheRVC.NotealsothattracesofRVCcaninhibitreversetranscriptases. Problem:Nospecificityisseeninthepull-downofmRNAs. Solution:Considerthefollowing: 1.Trytousehighsalt(1Mureaor300mMKAc)orheparininthewashbufferwhenperformingStep25. 2.MakesurethatthePCRreactionisstillinthelinearrangeattheendofthesecondaryPCR(the0.5-µLand1.5-µLinputconcentrationsshouldshowathreefolddifferenceinintensityontheDNAgel).IfthePCRreactionhasbeenexhausted,differencesbetweenspecificandnonspecificIPmightbelost. DISCUSSION ThemethoddescribedhereallowsfortheinvestigationofRNA-proteincomplexesandidentificationofmRNAtargetsofRNA-bindingproteinsinvivo.Althoughthemethodisgenerallyapplicable,itutilizeswhole-wormextractsasastartingmaterialfortheco-IPprocedure.Wormsareprotectedbyatoughcuticle,whichmakesitdifficulttoobtainintactextractsofhighproteinconcentration.Wehavetestedalternativeproceduresforhomogenizingworms,suchassonicationandtheuseofaFrenchpress.Inourhands,grindingfrozenwormpearlswithamortarandpestlegivesthemostreproducibleresultsandcoststheleast.DuringsonicationandFrenchpressing,localheatingofthesampleswasmeasured;thiscanaffectthequalityofthemRNA.Furthermore,thesemethodsrequirelargervolumesforefficientwormlysis,whicheitherreducestheproteinconcentrationoftheextractorincreasesthequantityofwormsneededforextractpreparation.Wealsoobservedthatitismorebeneficialforgermlineprotein/RNAcomplexestogrowthewormsonsolidsupportthaninliquidculture,wherethemorphologyofthegermlineandtheProgenyproducedareoflowerquality.Theuseofeggplatesisnotrecommended,becauseitisratherdifficulttoreducethehighproteinandlipidbackground,whichisaresultofcontaminatingchickenyolk. Goodcontrolsarecrucialforameaningfulresultinco-IPexperiments.Firstofall,itisimportanttouseanonspecificantibodyinparalleltothespecificantibodyasanegativecontrolfortheIP.AnexcellentnegativecontrolistheinclusionofanIPsamplefromawormextract(e.g.,frommutantanimals)thatdoesnotcontaintheproteinpulleddownfromthewild-typewormextract.However,makesuretoadjustthewild-typeandmutantextractstothesametotalproteinconcentrationpriortothepull-downreaction.Second,inthegene-specificPCR,itisimportanttotestforanabundantmRNAthatisnotexpectedtobeassociatedwiththeimmunoprecipitatedproteincomplex.SuchanmRNAservesasagoodspecificitycontrol,becauseitshouldeithernotbeenrichedinthespecificantibodysampleoverthenegativecontrolornotbepresentintheIPsamplesatall. Themethoddescribedhereprovidesatooltoexamineprotein-RNAcomplexcompositionsinvivo.However,itshouldalwaysbekeptinmindthattheprotein-RNAinteractionsidentifiedmaybemediatedbyotherproteins.Evenmoreimportantly,itmustberememberedthatapositiveco-IPresultdoesnotconfirmthatagivenprotein-RNAcomplexisaconsequenceofadirectphysicalinteractionbetweentheproteininquestionandthetestedmRNAmolecule. 您的称呼： 单位名称： 您的邮箱： 您的电话： 品牌与型号： 其他要求： 新闻动态 行业前沿 技术文章 最新产品 Surgiwax 巴西*蜡套装 4 盎司(约 113 克) 1包: 亚马逊中国: 化妆 2021-03-24 美国公司 Surgistar厂家 2019-08-28 美国公司 Surgistar产品 2020-10-13 进口试剂直采 Surgistar价格 2021-09-13 美国公司 一级代理 2018-07-16 抗体，试剂，血清，细胞 Surgistar授权 2018-06-17 一级代理 Surgistar官网 2021-09-09 中国公司 试剂产品信息 2018-09-15 《生命科学》【价格目录书评正版】_中图网 2021-02-01 原创:深圳生命科学价格优惠 Surgistar官网,是正品吗? 2019-01-10 有谁知道:深圳品牌试剂实验代理 Surgistar,有促销打折? 2020-08-22 有谁知道:深圳品牌试剂实验代理 Surgistar,有促销打折? 2020-08-22 求教:上海中国采购 Surgistar产品,求推荐! 2020-04-17 求解答:银川试剂采购 Surgistar产品,有谁知道吗? 2019-05-07 原创:深圳生命科学价格优惠 Surgistar官网,是正品吗? 2019-01-10 科普:西安中国采购 Surgistar,有优惠活动吗? 2018-08-04 shRNA干扰载体构建_白鹤翠鸣 2021-09-18 shRNA干扰载体构建_白鹤翠鸣 2021-08-31 动物肝脏中DNA的提取及检测实验报告 2021-09-10 Science子刊两篇论文带来阿尔茨海默症治疗新思路 医疗健康专区... 2021-08-29 阿尔茨海默病早期治疗有新思路 2021-08-30 恩他卡朋促进大鼠cAMP依赖的Cl分泌引起胃肠不适 实验方法 丁 ... 2021-09-17 COX2shRNA干扰载体的构建.pdf 2021-09-20 动物肝脏中DNA的提取及检测实验报告 2021-09-04 Surgistar/175005/10/175005 2026-07-06 Surgistar/962858/6/962858 2026-07-06 苏吉星/340850/1/340850 2026-07-06 Surgistar/165147/10/165147 2026-07-06 苏吉星/964501/6/964501 2026-07-06 Surgistar/Sharp全半径和侧刃，双斜面/S-6900 2026-07-06 苏尔吉斯塔/125029/10/125029 2026-07-06 苏吉星/125037/10/125037 2026-07-06 Copyright@苏州蚂蚁淘生物科技有限公司 2003-2021  188进口试剂采购网版权所有     苏ICP备17049038号-9 友情链接：  其他 188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap