Ⅰ. Ongera ibyiyumvo bya sisitemu yo kwitwara:
1. Tandukanya urwego rwo hejuru RNA:
Intsinzi ya cDNA igenda ituruka murwego rwo hejuru RNA.RNA-yuzuye cyane igomba kwemeza byibura igihe kirekire kandi ntigizwe na inhibitor zidafite imisemburo yo gufata amajwi, nka EDTA cyangwa SDS.Ubwiza bwa RNA bugena agaciro ntarengwa kamakuru yamakuru ushobora kwandukura kuri cDNA.Uburyo rusange bwo kweza RNA nuburyo bwintambwe yo gukoresha isoocyanate / acideophenol.Mu rwego rwo gukumira umwanda wa RNase, RNA yatandukanijwe n’icyitegererezo gikungahaye kuri RNase (nka pancreas) isaba kubika fordehide kugirango ibike RNA yo mu rwego rwo hejuru, ndetse ikaba ari myinshi cyane kubikwa igihe kirekire.RNA yakuwe mu mwijima w'imbeba ahanini yangiritse nyuma y'icyumweru kimwe kibitswe mu mazi, mu gihe RNA yakuwe mu mbeba y'imbeba yagumye ihagaze neza nyuma y'imyaka itatu ibitswe mu mazi.Mubyongeyeho, inyandiko-mvugo irenze 4kb irumva neza gukurikirana iyangirika rya RNase kuruta inyandiko nto.Kugirango hongerwe imbaraga mububiko bwa RNA icyitegererezo, RNA irashobora gushonga muri methalmamine ya ion, ikabikwa -70 ° C.Thylide ikoreshwa mukuzigama RNA ntigomba kuba irimo ibintu bitandukanye bitesha agaciro RNA.RNA, ikomoka kuri pancreas, irashobora gukizwa muri methalmamine byibuze umwaka.Mugihe witeguye gukoresha RNA, urashobora gukoresha uburyo bukurikira kugirango ugabanye RNA: ongeramo NaCl kuri 0.2m ninshuro 4 zubunini bwa Ethanol, shyira ubushyuhe bwicyumba muminota 3-5, na 10,000 × g centrifugal muminota 5.
2. Koresha inyandiko zinyuranye zidafite ibikorwa bya RNaseH (RNaseH-) ：
Inhibitori ya RNase ikunze kongerwaho kugirango ihindure transcript kugirango yongere uburebure numusaruro wa cDNA synthesis.Inhibitori ya RNase yongewemo mumurongo wambere wa synthesis reaction imbere ya buffers no kugabanya ibintu nka DTT kuko inzira ya synthesis ya pre-cDNA yerekana inhibitor, bityo ikarekura RNase iboshye itesha agaciro RNA.Protein RNase inhibitor irinda gusa kwangirika kwa RNA na RNase A, B, C, kandi ntibibuza RNase kuruhu, bityo rero hagomba kwitonderwa kutamenyekanisha RNase kurutoki nubwo hakoreshwa izo inhibitor.
Guhindura inyandiko-mvugo itera guhindura RNA muri cDNA.M-MLV na AMV zombi zifite ibikorwa bya endogenous RNaseH byiyongera kubikorwa byabo bya polymerase.Igikorwa cya RNaseH gihanganye nibikorwa bya polymerase kumurongo wa heterozygous wakozwe hagati yicyitegererezo cya RNA na primers ya ADN cyangwa umugozi wa cDNA, kandi ugatesha agaciro RNA: imirongo ya RNA mubice bya ADN.Inyandikorugero za RNA zateshejwe agaciro nigikorwa cya RNaseH ntizishobora gukoreshwa nkibisobanuro bifatika bya synthesis ya cDNA, bigabanya umusaruro nuburebure bwa cDNA.Kurandura cyangwa kugabanya cyane ibikorwa bya RNaseH bya reverse transcriptase byagira akamaro kanini.
SuperScriptⅡ ihinduranya inyandiko, MMLV ihinduranya inyandiko ya RNaseH- na thermoScript revers transcriptase, AMV ya RNaseH- yatanze cDNA ndende yuzuye kuruta MMLV na AMV.RT-PCR ibyiyumvo byatewe nubunini bwa cDNA ikomatanya.ThermoScript irumva cyane kuruta AMV.Ingano y'ibicuruzwa bya RT-PCR igarukira kubushobozi bwo guhinduranya inyandiko-mvugo yo guhuza cDNA, cyane cyane iyo ikoronije Cdnas nini.Ugereranije na MMLV, SuperScripⅡ yongereye cyane umusaruro wibicuruzwa birebire bya RT-PCR.RNaseH-'s reverse transcriptase nayo yongerera ubushyuhe ubushyuhe, bityo reaction irashobora gukorwa mubushyuhe burenze busanzwe bwa 37-42 ℃.Mugihe cyateganijwe cyo gusanisha, hakoreshejwe oligo (dT) primers na 10μCi [alpha-p] dCTP.Umusaruro wose wumunyururu wambere wabazwe ukoresheje uburyo bwimvura ya TCA.Uburebure bwuzuye cDNA bwasesenguwe hakoreshejwe ubunini bwakuweho no kubara muri gel ya alkaline agarose.
3. Ongera ubushyuhe bwo kubika ubushyuhe bwo kwandukura ：
Ubushyuhe bwo hejuru bufasha gufungura imiterere ya kabiri ya RNA no kongera umusaruro wa reaction.Kuri templates nyinshi za RNA, gufata RNA na primer kuri 65 ° C idafite buffer cyangwa umunyu hanyuma ukayikonjesha vuba kurubura bikuraho ibyiciro byinshi byisumbuye kandi bituma primers ihuza.Nyamara, inyandikorugero zimwe ziracyafite imiterere ya kabiri, nubwo nyuma yubushyuhe bwumuriro.Amplification yizi nyandikorugero zigoye zirashobora gukorwa hifashishijwe ThermoScript reverse transcriptase no gushyira reaction transcriptase reaction kubushyuhe bwo hejuru kugirango tunonosore amplification.Ubushyuhe bwo hejuru burashobora kandi kongera umwihariko, cyane cyane mugihe cDNA synthesis ikorwa hakoreshejwe primers yihariye (GSPS) (reba Umutwe wa 3).Niba ukoresha GSP, menya neza ko Tm agaciro ka primer ari kimwe nubushakashatsi buteganijwe.Ntukoreshe oligo (dT) na primers zidasanzwe hejuru ya 60 ℃.Ibisanzwe bisanzwe bigomba gufatwa kuri 25 ℃ muminota 10 mbere yo kwiyongera kuri 60 ℃.Usibye gukoresha ubushyuhe bwo hejuru bwo kwandukura, umwihariko urashobora kunozwa no kwimura mu buryo butaziguye ivangwa rya RNA / primer kuva kuri 65 ℃ gutandukanya ubushyuhe kuri transcription transcription ifite ubushyuhe no kongeramo ubushyuhe bwa 2 × reaction (cDNA yubushyuhe bwo gutangiza ubushyuhe).Ubu buryo bufasha gukumira guhuza intermolecular bibaho kubushyuhe buke.Gukoresha igikoresho cya PCR byoroshya ubushyuhe bwinshi busabwa kuri RT-PCR.
Ubushyuhe bwa Tth polymerase ikora nka polymerase ya ADN imbere ya Mg2 + na RNA polymerase imbere ya Mn2 +.Irashobora gufata ubushyuhe bugera kuri 65 ℃.Ariko, kuba Mn2 + mugihe cya PCR bigabanya ubudahemuka, bigatuma Tth polymerase idakwiriye gukwirakwizwa neza, nka clon ya cDNA.Byongeye kandi, Tth ntabwo ikora neza muguhindura transcription, igabanya sensibilité, kandi kubera ko enzyme imwe ishobora gukora transcription transcription na PCR, reaction yo kugenzura idafite transcription ntishobora gukoreshwa mugutandukanya ibicuruzwa byongerewe imbaraga bya cDNA nibya ADN byanduye.
4. Inyongera iteza imbere transcript reverse
Kwiyongera kwinyongeramusaruro, harimo glycerine na DMSO, kumurongo wambere wa synthesis reaction irashobora kugabanya ituze rya acide nucleic acide inshuro ebyiri kandi ikanakuraho imiterere ya kabiri ya RNA.Kugera kuri 20% glycerine cyangwa 10% DMSO irashobora kongerwaho bitagize ingaruka kubikorwa bya SuperScriptⅡ cyangwa MMLV.AMV irashobora kandi kwihanganira glycerol igera kuri 20% itagabanije ibikorwa.Kugirango urusheho kwiyumvamo RT-PCR muri SuperScriptⅡ reaction transcription reaction, 10% glycerol irashobora kongerwamo no gukingirwa kuri 45 ℃.Niba 1/10 cyibicuruzwa bya retrotranscription-reaction byongewe kuri PCR, kwibumbira hamwe kwa glycerol muri reaction ya amplification ni 0.4%, ntibihagije kubuza PCR.
5. Gutunganya RNaseH ：
Sensitivity irashobora kunozwa mugukemura cDNA synthesis reaction hamwe na RNaseH mbere ya PCR.Kuri templates zimwe, biratekerezwa ko RNA mumikorere ya cDNA synthesis irinda guhuza ibicuruzwa byongerewe imbaraga, mugihe ubuvuzi bwa RNaseH bushobora kongera sensibilité.Mubisanzwe, ubuvuzi bwa RNaseH burakenewe kugirango hongerwe uburebure buringaniye burebure bwa cDNA yerekana icyitegererezo, nka tuberous scheroseⅡ hamwe na kopi nke.Kuri iyi nyandikorugero igoye, RNaseH yazamuye ibimenyetso byakozwe na cDNA ikomatanya na SuperScriptⅡ cyangwa AMV.Kubisubizo byinshi bya RT-PCR, kuvura RNaseH birashoboka kuko intambwe ya 95 ℃ yandujwe na PCR isanzwe itanga hydrolyzes RNA kuva RNA: ikigo cya ADN.
6. Uburyo bunoze bwo kumenya umubare muto wa RNA ：
RT-PCR iragoye cyane mugihe haboneka bike bya RNA.Kwiyongera kwa glycogene nkumutwara mugihe cyo gutandukana kwa RNA bifasha kongera umusaruro wintangarugero nto.Glycogene idafite RNase irashobora kongerwaho mugihe kimwe na Trizol.Glycogene irashobora gushonga amazi kandi irashobora kuguma mugice cyamazi hamwe na RNA kugirango ifashe imvura ikurikira.Icyifuzo cyo kwibanda kuri glycogene idafite RNase ni 250μg / ml kuburugero ruri munsi ya 50mg ya tissue cyangwa selile 106 zifite imico.
Kwiyongera kwa acetylated BSA kugirango uhindure inyandiko zandikirwa ukoresheje SuperScriptⅡ birashobora kongera ibyiyumvo, kandi kuri RNA nkeya, kugabanya umubare wa SuperScriptⅡ no kongeramo ibice 40 bya RnaseOut nuclease inhibitor birashobora kuzamura urwego rwo gutahura.Niba glycogene ikoreshwa mugutandukanya RNA, hongerwaho BSA cyangwa RNase inhibitor kugirango uhindure reaction ukoresheje SuperScriptⅡ biracyasabwa.
Ⅱ. Ongera umwihariko wa RT-PCR
1. cNDA synthesis ：
Uburyo butatu butandukanye burashobora gukoreshwa mugutangiza umurongo wa mbere wa cDNA, kandi ugereranije umwihariko wa buri buryo bigira ingaruka kumubare n'ubwoko bwa cDNA ikomatanya.
Uburyo bwa primer busanzwe nuburyo buto bwihariye muburyo butatu.Primers ihujwe kurubuga rwinshi mumyandikire kugirango itange uburebure, igice cDNA.Ubu buryo bukoreshwa muburyo bwo kubona 5 ′ itondekanya hamwe na cDNA kuva mubishusho bya RNA hamwe n'uturere twa kabiri twubatswe cyangwa hamwe nibibuga bisoza inyandiko zinyuranye zidashobora kwigana.Kugirango ubone cDNA ndende, igipimo cya primers na RNA muri buri cyitegererezo cya RNA kigomba kugenwa muburyo bwiza.Intangiriro yibanze ya primers idasanzwe kuva kuri 50 kugeza 250ng kuri sisitemu ya reaction ya 20μl.Kuberako cDNA ikomatanya kuva RNA yose ukoresheje primers zidasanzwe ni ahanini ribosomal RNA, poly (A) + RNA muri rusange yatoranijwe nkicyitegererezo.
Oligo (dT) gutangira birasobanutse kuruta primers zidasanzwe.Ihuza umurizo wa poly (A) iboneka kuri 3 ′ iherezo rya mRNA muri selile nyinshi za eukaryotic.Kuberako poly (A) + RNA igera kuri 1% kugeza kuri 2% ya RNA yose, ingano nuburemere bwa cDNA ni bike cyane ugereranije niba hakoreshejwe primers zidasanzwe.Kubera umwihariko wacyo, oligo (dT) mubisanzwe ntabwo isaba optimizasiyo ya RNA kuri primer ratio 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 kandi ikwiranye nubushyuhe bwo hejuru.
Gene yihariye primers (GSP) ninziza nziza yibanze yintambwe yo kwandukura.GSP ni antisense oligonucleoside ishobora guhuza cyane na RNA ikurikirana, aho guhuza Rnas zose nka primers zidasanzwe cyangwa oligo (dT).Amategeko akoreshwa mugushushanya primers ya PCR nayo akoreshwa mugushushanya reaction ya reaction ya GSP.GSP irashobora kuba ikurikiranye kimwe na amplification primer yometse kumpera ya mRNA3 ′, cyangwa GSP irashobora gushushanywa kugirango igabanuke hepfo hamwe na primer amplification primer.Kubintu bimwe byongerewe imbaraga, birakenewe gushushanya primer zirenze imwe antisense primer kugirango RT-PCR igende neza kuko imiterere ya kabiri yintego RNA ishobora kubuza primer guhambira.Birasabwa gukoresha 1pmol antisense GSP muri sisitemu yambere ya synthesis reaction ya 20μl.
2. Ongera ubushyuhe bwo kubika ubushyuhe bwo kwandukura reverse
Kugirango ukoreshe byimazeyo GSP yihariye, hagomba gukoreshwa inyandiko-mvugo ihindagurika hamwe nubushyuhe bwo hejuru bwumuriro.Ubushyuhe butajegajega bushobora kwandikirwa ubushyuhe bwinshi kugirango byongere imbaraga.Kurugero, niba GSP ihujwe kuri 55 ° C, noneho umwihariko wa GSP ntukoreshwa neza niba transcript ihindurwa ikorwa kuri 37 ° C hamwe na buke buke ukoresheje AMV cyangwa M-MLV.Nyamara, SuperScripⅡ na ThermoScript irashobora kwitwara kuri 50 ℃ cyangwa irenga, ikuraho ibicuruzwa bidafite akamaro byakozwe mubushyuhe buke.Kubisobanuro byihariye, imvange ya RNA / primer irashobora kwimurwa biturutse ku bushyuhe bwa 65 ℃ gutandukana kugeza kuri transcription transcription ifata ubushyuhe hiyongereyeho 2 x ivanze ryashushe (gutangiza ubushyuhe bwa cDNA synthesis).Ibi bifasha gukumira guhuza hagati ya molekile ku bushyuhe buke.Gukoresha igikoresho cya PCR byoroshya ubushyuhe bwinshi busabwa kuri RT-PCR.
3. Kugabanya ADN yanduye ：
Imwe mu ngorane zishobora guterwa na RT-PCR nuko RNA yanduza ADN genomic.Gukoresha uburyo bwiza bwo gutandukanya RNA, nka Trizol Reagent, bigabanya kwanduza ADN genomic mumyiteguro ya RNA.Kugira ngo wirinde ibicuruzwa biva muri ADN genomic, RNA irashobora kuvurwa hamwe na DnasⅠ yo mu rwego rwo gukuraho ADN yanduye mbere yo kwandukura.Ingero zabitswe kuri 65 ℃ muri 2.0mM EDTA kuminota 10 kugirango ihagarike igogorwa rya DNaseⅠ.EDTA ikonjesha ion ya magnesium kugirango irinde magnesium ion iterwa na RNA hydrolysis ibaho mubushyuhe bwinshi.
Kugirango utandukanye cDNA yongerewe imbaraga na ADN yongerewe imbaraga za ADN, primers anneal ukwayo hamwe na exon yatandukanijwe irashobora gutegurwa.Ibicuruzwa bya PCR bikomoka kuri cDNA bizaba bigufi kuruta ibikomoka kuri ADN yanduye.Ubushakashatsi bugenzurwa butabanje kwandukura nabwo bukorwa kuri buri cyitegererezo cya RNA kugirango hamenyekane niba igice cyatanzwe kiva muri ADN genomic cyangwa cDNA.Ibicuruzwa bya PCR byabonetse mugihe hatabayeho kwandukura ibintu biva muri genome.
Ibicuruzwa bifitanye isano
RT-PCR BiroroshyeᵀᴹI (Intambwe imwe)
-Ibikoresho byintambwe imwe ituma guhinduranya transcription na PCR bikorerwa mumiyoboro imwe.Irakeneye gusa kongeramo inyandikorugero RNA, progaramu yihariye ya PCR na ddH yubusa ya RNase2O.
-Isesengura-nyaryo ryuzuye rya RNA rirashobora gukorwa vuba kandi neza.
-Igikoresho gikoresha uburyo bwihariye bwa Foregene reverse transcription reagent na Foregene HotStar Taq ADN Polymerase ihujwe na sisitemu idasanzwe yo gukora kugirango tunoze neza imikorere ya amplification hamwe nibisobanuro bya reaction.
-Uburyo bwiza bwo gukora butuma reaction igira sensibilité yo hejuru, gukomera kwumuriro, no kwihanganira neza.
-Ubushobozi buhagije bwo gukuraho gDNA, ishobora gukuraho gDNA mubishusho muminota 2.
-Uburyo bwiza bwo kwandukura sisitemu, bifata iminota 15 gusa kugirango urangize synthesis yumurongo wambere cDNA.
-Icyitegererezo cyoroshye: inyandikorugero zirimo ibintu byinshi bya GC hamwe nuburyo bugoye bwa kabiri birashobora kandi guhindurwa hamwe nubushobozi buhanitse.
-Ibihe byinshi-byunvikana sisitemu yo kwandukura, pg-urwego rwicyitegererezo nacyo gishobora kubona cDNA nziza.
-Uburyo bwo kwandukura sisitemu ifite ubushyuhe buhanitse, ubushyuhe bwiza bwa reaction ni 42 ℃, kandi buracyafite imikorere myiza yo kwandukura kuri 50 ℃.
Igihe cyo kohereza: Werurwe-07-2023
