一 Ongera ibyiyumvo bya sisitemu ya reaction ：

1. Gutandukanya RNA nziza cyane ：

Intsinzi ya cDNA ituruka muburyo bwiza bwa RNA.RNA yo mu rwego rwo hejuru igomba nibura kuba ndende-yuzuye kandi idafite inzitizi zinyuranye za transitase nka EDTA cyangwa SDS.Ubwiza bwa RNA bugena umubare ntarengwa wamakuru ukurikirana ushobora kwandukura muri cDNA.Uburyo busanzwe bwo kweza RNA nuburyo bumwe bwintambwe ukoresheje guanidine isothiocyanate / aside fenol.Kugira ngo wirinde kwanduzwa na RNase nyinshi, RNA yitandukanije n’intangarugero zikungahaye kuri RNase (nka pancreas) igomba kubikwa muri fordehide kugirango ibungabunge RNA nziza cyane cyane kubikwa igihe kirekire.RNA yakuwe mu mwijima w'imbeba ahanini yangiritse nyuma yo kubikwa mu mazi icyumweru kimwe, mu gihe RNA yakuwe mu mbeba y’imbeba yagumye ihagaze neza nyuma yo kubikwa mu mazi imyaka 3.Mubyongeyeho, inyandiko-mvugo irenze 4 kb yunvikana no guteshwa agaciro na RNase kuruta inyandiko nto.Kugirango hongerwe imbaraga za RNA zabitswe, RNA irashobora gushonga muri formamide deionion kandi ikabikwa kuri 70 ° C.Formamide ikoreshwa mu kubungabunga RNA igomba kuba idafite imyanda yangiza RNA.RNA iva pancreas irashobora kubikwa muri formamide byibuze umwaka.Mugihe witegura gukoresha RNA, urashobora gukoresha uburyo bukurikira kugirango ugabanye RNA: ongeramo NaCl kuri 0.2M ninshuro 4 zubunini bwa Ethanol, shyira mubushyuhe bwicyumba muminota 3-5, na centrifuge kuri 10,000 × g muminota 5.

2. Koresha RNaseH idakora (RNaseH-) inyandikomvugo reverse

Inhibitori ya RNase ikunze kongerwaho kugirango ihindure transcript kugirango yongere uburebure numusaruro wa cDNA synthesis.Inhibitori ya RNase igomba kongerwamo mugihe cyambere cya synthesis reaction imbere ya buffer na agent igabanya (nka DTT), kubera ko inzira yabanjirije synthesis ya cDNA yerekana inhibitor, bityo ikarekura RNase iboshye ishobora gutesha agaciro RNA.Intungamubiri za poroteyine RNase zirinda gusa kwangirika kwa RNA na RNase A, B, C, kandi ntukabuze RNase kuruhu, bityo rero wirinde kutamenyekanisha RNase mu ntoki zawe nubwo hakoreshejwe izo inhibitor.

Guhindura inyandiko-mvugo itera guhindura RNA kuri cDNA.M-MLV na AMV zombi zifite ibikorwa bya endogenous RNaseH byiyongera kubikorwa byabo bya polymerase.Igikorwa cya RNaseH nibikorwa bya polymerase birahatana hagati yumurongo wa Hybrid wakozwe hagati yicyitegererezo cya RNA na ADN primer cyangwa umugozi wagutse wa cDNA, kandi ugatesha agaciro umurongo wa RNA muri RNA: ikigo cya ADN.Inyandikorugero ya RNA yangijwe nigikorwa cya RNaseH ntigishobora kuba insimburangingo ifatika ya cDNA synthesis, igabanya umusaruro nuburebure bwa cDNA.Kubwibyo, byaba byiza gukuraho cyangwa kugabanya cyane ibikorwa bya RNaseH bya transcript transcript .。

SuperScript Ⅱ reverse transcriptase, RNaseH- MMLV reverse transcriptase hamwe na thermoScript revers transcriptase, RNaseH- AMV, irashobora kubona amafaranga menshi nuburebure bwuzuye cDNA kuruta MMLV na AMV.RT-PCR ibyiyumvo bizagira ingaruka kumubare wa cDNA.ThermoScript irumva cyane kuruta AMV.Ingano yibicuruzwa bya RT-PCR bigarukira kubushobozi bwa reaction transcriptase yo guhuza cDNA, cyane cyane iyo ikoronije cDNA nini.Ugereranije na MMLV, SuperScripⅡ yongereye cyane umusaruro wibicuruzwa birebire bya RT-PCR.RNaseH-reverse transcriptase nayo yongereye ubushyuhe, bityo reaction irashobora gukorwa mubushyuhe burenze 37-42 ° C.Mugihe cyifuzo cya synthesis, koresha oligo (dT) primer na 10 μCi ya [α-P] dCTP.Umusaruro rusange wumurongo wambere wabazwe ukoresheje uburyo bwimvura ya TCA.Uburebure bwa cDNA bwasesenguwe hakoreshejwe ingano-itondekanya ingano yacukuwe kandi ibarwa kuri alkaline agarose gel.

3. Kuzamura ubushyuhe bwa incubation kugirango uhindure transcript ：

Ubushyuhe bwo hejuru bwa incubation bufasha gufungura imiterere ya RNA ya kabiri, byongera umusaruro wa reaction.Kuri templates nyinshi za RNA, gushiramo RNA na primers kuri 65 ° C idafite buffer cyangwa umunyu, hanyuma gukonjeshwa byihuse kurubura bizakuraho ibyubatswe byombi kandi bizemerera primers guhuza.Nyamara, inyandikorugero zimwe ziracyafite ibyiciro bya kabiri, nubwo nyuma yubushyuhe.Amplification yizi nyandikorugero zitoroshye zirashobora gukorwa hifashishijwe ThermoScript Reverse Transcriptase hanyuma ugashyira reaction ya reaction ya reaction ku bushyuhe bwo hejuru kugirango tunonosore amplification.Ubushyuhe bwo hejuru bwa incubation burashobora kandi kongera umwihariko, cyane cyane iyo primers yihariye ya gen (GSP) ikoreshwa muguhindura cDNA (reba Umutwe 3).Niba ukoresha GSP, menya neza ko Tm ya primers ari kimwe nubushyuhe buteganijwe.Ntukoreshe oligo (dT) na primers zidasanzwe hejuru ya 60 ° C.Ibisanzwe bisanzwe bisaba incububasi kuri 25 ° C muminota 10 mbere yo kwiyongera kuri 60 ° C.Usibye gukoresha ubushyuhe bwo hejuru bwo kwandukura, umwihariko urashobora kandi kunozwa no kwimura mu buryo butaziguye ivangwa rya RNA / primer kuva ku bushyuhe bwa 65 ° C ku bushyuhe bwa reaction ya incubation hanyuma ukongeramo ubushyuhe bwa 2 × reaction (cDNA ishyushye-itangira synthesis).Ubu buryo bufasha gukumira guhuza intermolecular bibaho kubushyuhe buke.Guhindura ubushyuhe bwinshi busabwa kuri RT-PCR birashobora koroshya ukoresheje cycler yumuriro.

Tth thermostable polymerase ikora nka polymerase ya ADN imbere ya Mg2 + kandi nka polymerase ya RNA imbere ya Mn2 +.Irashobora kugumana ubushyuhe ku bushyuhe ntarengwa bwa 65 ° C.Ariko, kuba Mn2 + mugihe cya PCR bigabanya ubudahemuka, bigatuma Tth polymerase idakwiriye gukwirakwizwa neza, nko gukoroniza cDNA.Byongeye kandi, Tth ifite ubushobozi buke bwo kwandukura inyandiko, bigabanya sensibilité, kandi, kubera ko transcription transcription na PCR bishobora gukorwa hamwe na enzyme imwe, reaction reaction idafite transcription ntishobora gukoreshwa mugereranya ibicuruzwa byongera cDNA hamwe na ADN yanduye.Ibicuruzwa byongerewe imbaraga byari bitandukanye.

4. Inyongera ziteza imbere transcript reverse

Inyongeramusaruro zirimo glycerol na DMSO zongewe kumurongo wa mbere wa synthesis reaction, ishobora kugabanya ituze rya acide nucleic acide kabiri kandi igahambura imiterere ya kabiri ya RNA.Kugera kuri 20% glycerol cyangwa 10% DMSO irashobora kongerwaho bitagize ingaruka kubikorwa bya SuperScript II cyangwa MMLV.AMV irashobora kandi kwihanganira glycerol igera kuri 20% nta gutakaza ibikorwa.Kugirango urusheho kwiyumvamo RT-PCR muri reaction ya SuperScriptⅡ ihinduranya inyandiko, glycerol 10% irashobora kongerwamo no gushyirwaho kuri 45 ° C.Niba 1/10 cyibicuruzwa biva mu mahanga byongewe kuri PCR, noneho kwibumbira hamwe kwa glycerol muri reaction ya amplification ni 0.4%, ntibihagije kubuza PCR.

5. Ubuvuzi bwa RNaseH ：

Kuvura cDNA synthesis reaction hamwe na RNaseH mbere ya PCR birashobora kongera sensibilité.Kuri templates zimwe, biratekerezwa ko RNA muri cDNA synthesis reaction ibuza guhuza ibicuruzwa byongera imbaraga, muribwo kuvura RNaseH bishobora kongera sensibilité.Mubisanzwe, kuvura RNaseH birakenewe mugihe wongereye uburebure bwuzuye bwa cDNA yerekana inyandikorugero, nka kopi ntoya ya tuber scherose II.Kuri iyi nyandikorugero igoye, kuvura RNaseH byongereye ibimenyetso byakozwe na SuperScript II cyangwa AMD-ikomatanya cDNA.Kubisubizo byinshi bya RT-PCR, kuvura RNaseH ntabwo byemewe, kubera ko intambwe yo gutandukanya PCR kuri 95 ° C muri rusange hydrolyzes RNA muri RNA: ikigo cya ADN.

6. Kunoza uburyo buto bwo kumenya RNA ：

RT-PCR iragoye cyane mugihe RNA nkeya gusa.Glycogen yongeyeho nkuwitwaye mugihe cya RNA yigunga ifasha kongera umusaruro wintangarugero nto.RNase idafite glycogene irashobora kongerwaho mugihe kimwe no kongeramo Trizol.Glycogene irashobora gushonga amazi kandi irashobora kubikwa mugice cyamazi hamwe na RNA kugirango ifashe imvura ikurikira.Kuburugero ruri munsi ya mg 50 ya tissue cyangwa selile 106 zifite imico, icyifuzo cyo kwibanda kuri glycogene idafite RNase ni 250 μg / ml.

Ongeramo acetylated BSA muburyo bwo guhindura inyandiko ukoresheje SuperScript II irashobora kongera ibyiyumvo, kandi kuri RNA nkeya, kugabanya umubare wa SuperScript II no kongeramo ibice 40 bya RNaseOut nuclease inhibitor bishobora kongera urwego rwo gutahura.Niba glycogene ikoreshwa mugikorwa cyo kwigunga kwa RNA, birasabwa kongeramo BSA cyangwa RNase inhibitor mugihe ukoresheje SuperScript II kugirango uhindure inyandiko.

二 Kongera umwihariko wa RT-PCR

1. CND Asynthesis ：

Igice cya mbere cDNA synthesis irashobora gutangizwa hakoreshejwe uburyo butatu butandukanye, ugereranije umwihariko wabyo ugira ingaruka kumubare n'ubwoko bwa cDNA ikomatanya.

Uburyo bwa primer random bwari buto cyane muburyo butatu.Primers anneal kurubuga rwinshi murinyandiko-mvugo, ibyara bigufi, uburebure bwa cDNAs.Ubu buryo bukoreshwa kenshi kugirango ubone 5 ′ kurangiza no kubona cDNA kuva mubishusho bya RNA hamwe n'uturere twubatswe kabiri cyangwa hamwe na site yo guhagarika idashobora kwiganwa na transcript transcriptase.Kugirango ubone cDNA ndende, igipimo cya primers na RNA muri buri cyitegererezo cya RNA kigomba kugenwa muburyo bwiza.Gutangira kwibanda kuri primers zidasanzwe kuva kuri 50 kugeza 250 ng kuri 20 μl reaction.Kubera ko cDNA ikomatanya kuva RNA yose ukoresheje primers zidasanzwe ni cyane cyane ribosomal RNA, poly (A) + RNA muri rusange ihitamo nkicyitegererezo.

Oligo (dT) primers zirasobanutse kuruta primers zidasanzwe.Ihuza umurizo wa poly (A) iboneka kuri 3 ′ iherezo rya mRNAs nyinshi.Kuberako poly (A) + RNA igera kuri 1% kugeza 2% ya RNA yose, ingano nuburemere bwa cDNA ni bike cyane ugereranije na primers zidasanzwe.Kubera umwihariko wacyo, oligo (dT) mubisanzwe ntabwo isaba guhuza igipimo cya RNA na primers na poly (A) + guhitamo.Birasabwa gukoresha 0.5μg oligo (dT) kuri sisitemu ya 20μl reaction.oligo (dT) 12-18 ikwiranye na RT-PCR.Sisitemu ya ThermoScript RT-PCR itanga oligo (dT) 20 kubera ubushyuhe bwiza bwumuriro kubushyuhe bwo hejuru.

Gene yihariye primers (GSP) niyo primers yihariye yintambwe yo kwandukura.GSP ni antisense oligonucleotide ishobora guhuza cyane cyane intego ya RNA ikurikirana, bitandukanye na primers zidasanzwe cyangwa oligo (dT), ihuza RNAs zose.Amategeko amwe akoreshwa mugushushanya primers ya PCR akoreshwa mugushushanya GSP muburyo bwo kwandukura.GSP irashobora kuba ikurikiranye kimwe na amplification primer ihuza 3′-iherezo rya mRNA, cyangwa GSP irashobora gushushanyirizwa kumurongo wo hasi wa reaction ya amplification primer.Kubintu bimwe byongeweho, primer zirenze imwe antisense primer igomba gutegurwa kugirango RT-PCR igende neza kuko imiterere ya kabiri yintego RNA ishobora kubuza guhuza primer.Birasabwa gukoresha 1 pmol antisense GSP muri 20 μl ya mbere ya synthesis reaction.

2. Kuzamura ubushyuhe bwa incubation kugirango uhindure transcript ：

Kugirango ukoreshe byuzuye inyungu zuzuye za GSP, hagomba gukoreshwa inyandiko-mvugo ihindagurika hamwe nubushyuhe bwo hejuru.Thermostable reverse transcriptase irashobora gushirwa mubushyuhe bwo hejuru kugirango yongere ubukana.Kurugero, niba GSP yometse kuri 55 ° C, umwihariko wa GSP ntuzakoreshwa neza niba AMV cyangwa M-MLV ikoreshwa muguhindura transcription kumurongo muke wa 37 ° C.Nyamara, SuperScript II na ThermoScript birashobora gukorerwa kuri 50 ° C cyangwa hejuru yayo, bizakuraho ibicuruzwa bidasanzwe byakozwe mubushyuhe buke.Kubisobanuro byihariye, kuvanga RNA / primer birashobora kwimurwa biturutse ku bushyuhe bwa 65 ° C kugeza ku bushyuhe bwa reaction ya incubation hanyuma bikongerwaho mbere yo gushyuha 2 × reaction ivanze (cDNA synthesis ishyushye itangira).Ibi bifasha kwirinda guhuza intermolecular guhuza ubushyuhe buke.Ubushyuhe bwinshi busabwa kuri RT-PCR burashobora koroshya ukoresheje icyuma gishyuha.

3. Kugabanya ADN yanduye ：

Ikibazo gishobora guhura na RT-PCR ni kwanduza ADN genomic muri RNA.Gukoresha uburyo bwiza bwo kwigunga bwa RNA, nka Trizol Reagent, bizagabanya umubare wa ADN genomic yanduza imyiteguro ya RNA.Kugira ngo wirinde ibicuruzwa biva muri ADN genomic, RNA irashobora kuvurwa hamwe na DNase ya amplification yo mu rwego rwo gukuraho ADN yanduye mbere yo kwandukura.Igogorwa rya DNase I ryarangijwe no gushiramo ingero muri 2.0 mM EDTA muminota 10 kuri 65 ° C.EDTA irashobora gushonga ioni ya magnesium, ikarinda magnesium ion iterwa na RNA hydrolysis mubushyuhe bwinshi.

Kugirango utandukane cDNA yongerewe kwanduza ibicuruzwa byongera ADN ya ADN, primers irashobora gushushanywa ko buri anneal kugirango itandukane exons.Ibicuruzwa bya PCR bikomoka kuri cDNA bizaba bigufi kuruta ibikomoka kuri ADN yanduye.Byongeye kandi, ubushakashatsi bwo kugenzura butabanje kwandukura bwakorewe kuri buri cyitegererezo cya RNA kugirango hamenyekane niba igice cyatanzwe cyakomotse kuri ADN genomic cyangwa cDNA.Ibicuruzwa bya PCR byabonetse nta transcription transcription biva muri genome.