一、 Wedzera kunzwisiswa kweiyo reaction system:

1. Isolate yemhando yepamusoro RNA:

Kubudirira cDNA synthesis kunobva kumhando yepamusoro RNA.Yepamusoro-soro RNA inofanira kunge izere-kureba uye isina reverse transcriptase inhibitors seEDTA kana SDS.Hunhu hweRNA hunotaridza huwandu hwehuwandu hwekutevedzana ruzivo rwaunogona kunyora mu cDNA.Nzira yakajairika yekuchenesa RNA inzira imwe-nhanho uchishandisa guanidine isothiocyanate/asidhi phenol.Kudzivirira kusvibiswa nekutevera huwandu hweRNase, RNA yakaparadzaniswa kubva kuRNase-rich samples (senge pancreas) inoda kuchengetwa mu formaldehyde kuchengetedza yemhando yepamusoro RNA, kunyanya yekuchengetedza kwenguva refu.Iyo RNA yakabviswa pachiropa chegonzo yakaderedzwa mushure mekuchengetwa mumvura kwevhiki imwe, nepo RNA yakatorwa kubva kumakonzo spleen yakaramba yakagadzikana mushure mekuchengetwa mumvura kwemakore matatu.Pamusoro pezvo, zvinyorwa zvinoreba kupfuura 4 kb zvinonyanya kunzwisiswa nekudzikisirwa nekutsvaga RNases pane zvinyorwa zvidiki.Kuti uwedzere kugadzikana kwemasamples eRNA akachengetwa, RNA inogona kunyungudika mu deionized formamide uye kuchengetwa pa -70 ° C.Formamide inoshandiswa kuchengetedza RNA inofanirwa kunge isina marara eRNA-anosvibisa.RNA kubva pancreas inogona kuchengetedzwa mu formamide kweinenge gore rimwe.Paunenge uchigadzirira kushandisa RNA, unogona kushandisa nzira inotevera yekunaya RNA: wedzera NaCl ku 0.2M uye 4 nguva yevhoriyamu ye ethanol, isa pamhepo tembiricha kwemaminitsi 3-5, uye centrifuge pa10,000 × g kwemaminitsi mashanu.

2. Shandisa iyo RNaseH-inactive (RNaseH-) reverse transcriptase:

RNase inhibitors inowanzowedzerwa kudzoreredza maitiro ekunyora kuti awedzere kureba uye goho re cDNA synthesis.RNase inhibitors inofanira kuwedzerwa panguva yekutanga-strand synthesis reaction muhupo hwebhafa uye inoderedza agent (yakadai seDTT), nokuti maitiro asati aitwa cDNA synthesis denatures inhibitor, nokudaro achisunungura RNase yakasungwa inogona kuderedza RNA.Protein RNase inhibitors inodzivirira chete kuparara kweRNA neRNase A, B, C, uye haidziviriri RNase paganda, saka chenjerera kuti usaunza RNase kubva paminwe yako pasinei nekushandiswa kweizvi inhibitors.

Reverse transcriptase inokonzeresa shanduko yeRNA kuita cDNA.Ose ari maviri M-MLV uye AMV ane endogenous RNaseH chiitiko mukuwedzera kune yavo yega polymerase chiitiko.Chiitiko cheRNaseH uye chiitiko chepolymerase chinokwikwidzana kune imwe hybrid strand yakaumbwa pakati peRNA template neDNA primer kana cDNA yekuwedzera strand, uye kusvibisa iyo RNA strand muRNA: DNA yakaoma.Iyo RNA template yakaderedzwa neRNaseH chiitiko haichakwanisa kushanda seyanoshanda substrate ye cDNA synthesis, iyo inoderedza goho uye kureba kwe cDNA synthesis.Naizvozvo, zvingave zvinobatsira kubvisa kana kuderedza zvakanyanya iyo RNaseH chiitiko che reverse transcriptase..

SuperScript Ⅱ reverse transcriptase, RNaseH- MMLV reverse transcriptase uye thermoScript reverse transcriptase, RNaseH- AMV, inogona kuwana huwandu uye hurefu hwakazara cDNA kupfuura MMLV ne AMV.RT-PCR senitivity ichakanganiswa nehuwandu hwe cDNA synthesis.ThermoScript inonyanya kunzwa kupfuura AMV.Saizi yezvigadzirwa zveRT-PCR inoganhurirwa nekugona reverse transcriptase kugadzira cDNA, kunyanya kana uchigadzira macDNA akakura.Kuenzaniswa neMMLV, SuperScripⅡ yakawedzera zvakanyanya goho rezvigadzirwa zveRT-PCR refu.Iyo RNaseH-reverse transcriptase zvakare yakawedzera kudziya kwekushisa, saka maitiro anogona kuitwa patembiricha yepamusoro pane yakajairika 37-42°C.Pasi pemamiriro akakurudzirwa ekugadzirisa, shandisa oligo (dT) primer uye 10 μCi ye [α-P] dCTP.Zvose zvibereko zvekutanga strand zvakaverengwa uchishandisa TCA precipitation method.Hurefu hwakazara cDNA yakaongororwa pachishandiswa saizi-yakarongwa mabhendi akachekwa uye akaverengerwa pane alkaline agarose gel.

3. Simudza tembiricha ye incubation yekureverse transcription:

Iyo yakakwirira incubation tembiricha inobatsira kuvhura iyo RNA yechipiri chimiro, ichiwedzera goho rekuita.Kune akawanda matemplate eRNA, incubating iyo RNA uye maprimers pa65 ° C isina buffer kana munyu, inoteverwa nekukurumidza kutonhora paaizi kunobvisa akawanda echipiri maumbirwo uye kubvumira maprimers kusunga.Nekudaro, mamwe matemplate achine zvimiro zvechipiri, kunyangwe mushure mekupisa denaturation.Kukwidziridzwa kweaya matemplate akaoma kunogona kuitwa uchishandisa ThermoScript Reverse Transcriptase uye nekuisa reverse transcript reaction pane tembiricha yepamusoro kuti iwedzere kukwidziridzwa.Kupisa kwepamusoro-soro kunogonawo kuwedzera hunhu, kunyanya kana gene-specific primers (GSP) ichishandiswa cDNA synthesis (ona Chitsauko 3).Kana ukashandisa GSP, ita shuwa kuti Tm yemaprimers yakafanana neinotarirwa tembiricha yekuisa.Usashandise oligo(dT) uye maprimers asina kurongeka ari pamusoro pe60°C.Maprimers asina kujairika anoda incubation pa25°C kwemaminetsi gumi asati awedzera kusvika pa60°C.Pamusoro pekushandisa yakakwira reverse transcript tembiricha, humbowo hunogona kuvandudzwa nekufambisa zvakananga RNA/primer musanganiswa kubva pa65°C denaturation tembiricha kuenda kune reverse transcription incubation tembiricha uye nekuwedzera pre-kudziirwa 2× reaction musanganiswa (cDNA inopisa-kutanga synthesis) .Iyi nzira inobatsira kudzivirira intermolecular base pairing inoitika pakudzika kwakadzika.Iyo yakawanda tembiricha switching inodiwa yeRT-PCR inogona kurerutswa nekushandisa inopisa cycler.

Tth thermostable polymerase inoshanda seDNA polymerase pamberi peMg2+ uye seRNA polymerase pamberi peMn2+.Inogona kuchengetwa ichidziya pakupisa kukuru kwe65°C.Zvisinei, kuvapo kweMn2 + panguva yePCR kunoderedza kuvimbika, izvo zvinoita kuti Tth polymerase isanyanya kukodzera kukwidziridzwa kwepamusoro-soro, zvakadai sekugadzirisa cDNA.Pamusoro pezvo, Tth ine yakaderera reverse transcription performance, iyo inoderedza kunzwisiswa, uye, sezvo reverse transcription uye PCR inogona kuitwa neimwe enzyme, kudzora maitiro pasina reverse transcription haigone kushandiswa kuenzanisa cDNA amplification zvigadzirwa nekusvibisa genomic DNA.Zvigadzirwa zveamplification zvakaparadzaniswa.

4. Zviwedzerwa zvinosimudzira reverse transcription:

Zviwedzerwa zvinosanganisira glycerol uye DMSO zvinowedzerwa kune yekutanga-strand synthesis reaction, iyo inogona kuderedza kugadzikana kweiyo nucleic acid kaviri-strand uye kusunungura yechipiri chimiro cheRNA.Kusvikira ku20% glycerol kana gumi muzana DMSO inogona kuwedzerwa pasina kukanganisa SuperScript II kana MMLV chiitiko.AMV inogonawo kushivirira kusvika ku20% glycerol pasina kurasikirwa kwebasa.Kuti uwedzere kunzwisiswa kweRT-PCR muSuperScriptⅡ reverse transcription reaction, 10% glycerol inogona kuwedzerwa nekuiswa pa45°C.Kana 1/10 ye reverse transcription reaction product yakawedzerwa kuPCR, ipapo kusungirirwa kweglycerol mukuita kweamplification ndeye 0.4%, iyo isina kukwana kuvharidzira PCR.

5. RNaseH kubata:

Kurapa kwe cDNA synthesis reactions neRNaseH pamberi pePCR kunogona kuwedzera kunzwa.Kune mamwe matemplate, zvinofungidzirwa kuti RNA mune cDNA synthesis reaction inodzivirira kusungwa kwezvigadzirwa zvekusimudzira, mune iyo nyaya RNaseH kurapwa kunogona kuwedzera kunzwa.Kazhinji, kurapwa kweRNaseH kunodiwa kana uchikudza matemplate akareba akazara-akazara cDNA, senge yakaderera-copy tuberous scheresis II.Kune iyi yakaoma template, kurapwa kweRNaseH kwakasimudzira chiratidzo chakagadzirwa neSuperScript II kana AMV-yakagadzirwa cDNA.Kune akawanda maRT-PCR maitiro, RNaseH kurapwa isarudzo, nekuti PCR denaturation nhanho pa95 ° C inowanzo hydrolyze iyo RNA muRNA: DNA yakaoma.

6. Kuvandudzwa kweDiki RNA Yekuona Nzira:

RT-PCR inonyanya kunetsa kana ishoma chete yeRNA iripo.Glycogen yakawedzerwa semutakuri panguva yekuzviparadzanisa neRNA inobatsira kuwedzera goho remasampuli madiki.RNase-isina glycogen inogona kuwedzerwa panguva imwe chete nekuwedzera Trizol.Glycogen inonyungudika mumvura uye inogona kuchengetwa muchikamu chemvura ine RNA kubatsira kunaya kunotevera.Kune samples isingasviki 50 mg yematishu kana 106 maseru akasimwa, iyo yakakurudzirwa kuunganidzwa kweRNase-isina glycogen ndeye 250 μg/ml.

Kuwedzera acetylated BSA kune reverse transcription reaction uchishandisa SuperScript II inogona kuwedzera kunzwisiswa, uye kune madiki eRNA, kuderedza huwandu hweSuperScript II uye kuwedzera makumi mana ezvikamu zveRNaseOut nuclease inhibitor inogona kuwedzera mwero wekuonekwa.Kana glycogen ikashandiswa muRNA yekuzviparadzanisa nevamwe, ichiri kukurudzirwa kuwedzera BSA kana RNase inhibitor paunenge uchishandisa SuperScript II yekudzosera reverse transcription reaction.

二、 Wedzera RT-PCR chaiyo

1. CND Asynthesis:

Chekutanga-strand cDNA synthesis inogona kutangwa uchishandisa nzira nhatu dzakasiyana, hukama hwehukama hunokanganisa huwandu uye rudzi rwecDNA yakagadzirwa.

Iyo isina kurongeka primer nzira yaive yakaderera pane nzira nhatu idzi.Primers anneal panzvimbo dzakawanda mukati mezvinyorwa zvese, zvichigadzira mapfupi, mashoma-kureba cDNAs.Iyi nzira inowanzoshandiswa kuwana 5 'magumo ekupedzisira uye kuwana cDNA kubva kuRNA templates ine nharaunda dzechipiri chimiro kana nenzvimbo dzekugumisa dzisingagoni kudzokororwa ne reverse transcriptase.Kuti uwane iyo yakareba cDNA, chiyero cheprimers kuRNA mune yega yega sampu yeRNA inoda kutariswa zvine simba.Iko kutanga kusungirirwa kwezvisina kurongeka zvekutanga zvakabva pa50 kusvika 250 ng pa20 μl reaction.Sezvo cDNA yakagadzirwa kubva kuRNA yakazara uchishandisa zvisina kurongeka primers inonyanya ribosomal RNA, poly(A)+RNA inowanzosarudzwa setemplate.

Oligo (dT) maprimers akanyanya kujeka pane zvakangoitika zvekutanga.Inosanganiswa kusvika papoly(A) muswe unowanikwa kumagumo e3′ eukaryotic mRNAs zhinji.Nekuti poly(A)+ RNA ingangoita 1% kusvika 2% yeRNA yakazara, huwandu uye kuomarara kwecDNA ishoma pane nematangiro asina kujairika.Nekuda kwehukuru hwayo hwepamusoro, oligo(dT) kazhinji haidi optimization yereshiyo yeRNA kune yekutanga uye poly(A)+ kusarudzwa.Inokurudzirwa kushandisa 0.5μg oligo(dT) pa20μl reaction system.oligo(dT)12-18 inokodzera akawanda RT-PCR.Iyo ThermoScript RT-PCR Sisitimu inopa oligo(dT)20 nekuda kwekugadzikana kwayo kurinani kwekupisa kwepamusoro incubation tembiricha.

Gene specific primers (GSP) ndiwo anonyanya kutarisisa eiyo reverse transcription nhanho.GSP is an antisense oligonucleotide iyo inogona kunyatso hybridize kune iyo RNA target sequence, kusiyana ne random primers kana oligo(dT), iyo anneal kune ese maRNA.Mitemo yakafanana inoshandiswa kugadzira PCR primers inoshanda kune dhizaini yeGSP mune reverse transcription reaction.Iyo GSP inogona kuve yakatevedzana seyekusimudza primer inoenda ku3′-yakanyanya kumagumo emRNA, kana GSP inogona kugadzirwa kudzika kudzika kweiyo reverse amplification primer.Kune zvimwe zvidzidzo zvakakwidziridzwa, inodarika imwe antisense primer inoda kugadzirwa kuti ibudirire RT-PCR nekuti chechipiri chimiro chechinangwa RNA chinogona kudzivirira primer kusunga.Zvinokurudzirwa kushandisa 1 pmol antisense GSP mune 20 μl yekutanga-strand synthesis reaction.

2. Simudza tembiricha ye incubation yekureverse transcription:

Kuti utore mukana wakazara weiyo yakazara mukana weGSP chaiyo, reverse transcriptase ine yakakwirira thermostability inofanira kushandiswa.Thermostable reverse transcriptases inogona kuiswa patembiricha yepamusoro kuti iwedzere kuita stringency.Semuyenzaniso, kana GSP ikawedzera pa55°C, humbowo hweGSP hauzoshandiswe zvizere kana AMV kana M-MLV ikashandiswa kunyora reverse pa37°C.Nekudaro, SuperScript II uye ThermoScript inogona kubatwa pa50 ° C kana kupfuura, izvo zvinobvisa zvisiri izvo zvigadzirwa zvinogadzirwa pakudziya kwakadzika.Nekunyanya kutaurwa, iyo RNA/primer musanganiswa inogona kutamiswa yakananga kubva pa65°C denaturation tembiricha kuenda kune reverse transcription incubation tembiricha uye yowedzerwa kune pre-kudziirwa 2× reaction musanganiswa (cDNA synthesis inopisa kutanga).Izvi zvinobatsira kudzivirira intermolecular base pairing pane yakaderera tembiricha.Iyo yakawanda tembiricha shanduko inodiwa yeRT-PCR inogona kurerutswa nekushandisa inopisa cycler.

3. Inoderedza genomic DNA kusvibiswa:

Chinhu chinogona kuoma chakasangana neRT-PCR ndiko kusvibiswa kwegenomic DNA muRNA.Kushandisa nzira yakanaka yeRNA yekuzviparadzanisa nevamwe, yakadai seTrizol Reagent, inoderedza huwandu hwejenomic DNA inosvibisa gadziriro yeRNA.Kuti udzivise zvigadzirwa zvinobva kugenomic DNA, RNA inogona kurapwa neamplification-giredhi DNase I kubvisa inosvibisa DNA isati yadzoserwa kunyorwa.Kugayiwa kweDNase I kwakagumiswa nekupinza masamples mu2.0 mM EDTA kwemaminetsi gumi pa65°C.EDTA inogona chelate magnesium ions, kudzivirira magnesium ion-inotsamira RNA hydrolysis pakupisa kwakanyanya.

Kuitira kuparadzanisa amplified cDNA kubva pakusvibisa genomic DNA amplification zvigadzirwa, maprimers anogona kugadzirwa kuti anneal yega yega iparadzanise maexons.PCR zvigadzirwa zvakatorwa kubva kucDNA zvichave zvipfupi pane izvo zvakatorwa kubva kune yakasvibiswa genomic DNA.Pamusoro pezvo, kuyedza kwekudzora pasina kudzoreredza kunyorwa kwakaitwa pane yega yega RNA template kuona kana chidimbu chakapihwa chakabva kugenomic DNA kana cDNA.Chigadzirwa chePCR chinowanikwa pasina reverse transcription chinobva kune genome.