Igeragezwa rya RT-qPCR ririmo gukuramo RNA no gusuzuma ubuziranenge, guhinduranya inyandiko na qPCR intambwe eshatu, buri ntambwe ifite ingamba nyinshi zo kwirinda, tuzabimenyesha birambuye hepfo.

Ⅰ.Isuzuma ryiza rya RNA

Mu igeragezwa rya RT-qPCR, nyuma yo kurangiza gukuramo RNA, hagomba gusuzumwa ubuziranenge bwa RNA, kandi ubushakashatsi bwo gukurikirana bushobora gukorwa nyuma yujuje ibyangombwa.Uburyo bwo gusuzuma burimo spekitifotometero, Agilent gel electrophorei, Agilent 2100 isesengura, muribwo buryo bukoreshwa cyane na spekitifotometero na agarose gel electrophorei.Twabibutsa ko ubu buryo bwombi bugomba gukoreshwa hamwe kugirango burangize gutahura no gusesengura kwibanda kwa RNA, ubuziranenge nubunyangamugayo, kugirango ubuziranenge bwa RNA bugerweho.

Bifitanye isano na RNA yo kwigunga: 

Akagari Igiteranyo cya RNA Igikoresho cyo kwigunga

RNA isukuye cyane kandi yujuje ubuziranenge RNA irashobora kuboneka muri selile zitandukanye zumuco muri 11min.

Inyamaswa Igiteranyo Cyuzuye RNA

Byihuse kandi neza gukuramo RNA-yuzuye kandi yuzuye-yuzuye muri RNA inyama zinyamaswa zitandukanye.

Ikirangantego:

Ikirangantego gikoreshwa cyane cyane kugirango hamenyekane ubunini bwa RNA nubuziranenge, ariko ntibishobora kumenya ubusugire bwa RNA nibisigisigi bya genomic.Muri byo, A260 / 280 na A260 / 230 ni ibipimo by'ingenzi mu gutahura isuku ya RNA, kandi isuku ya RNA irashobora kumenyekana ukurikije ihindagurika ry'agaciro kabo:

1. 1.9 2.1, byerekana ko bishoboka kwangirika kwa RNA igice, gishobora kwemezwa na agarose gel electrophorei.

2. 2.0

Agarose gel electrophorei:

Agarose gel electrophoresis assay irashobora gusesengura ubunyangamugayo bwa RNA, ibisigisigi bya genome na proteyine, ariko ntibishobora kugereranya neza ubunini bwa RNA cyangwa kumenya ibisigisigi bya reagent.Fata urugero rwa eukaryotic RNA urugero:

1. RNA yakorewe agarose gel electrophorei.Niba hari imirongo itatu yonyine ya 28sRNA, 18sRNA na 5.8sRNA kurikarita ya gel, byerekana ko RNA yakuweho idahwitse.Niba hari ikintu gikurura, cyerekana kwangirika kwa RNA igice.

2. Niba hari umurongo umwe urumuri hagati yumwobo wa kole na 28sRNA, hashobora kubaho ibisigisigi bya ADN.

3. Niba amabandi agaragara mu mwobo wa kole, byerekana ko hashobora kubaho ibisigazwa bya poroteyine nibindi bintu bya macromolekula.

Ⅱ. Guhindura inyandiko

Gukuramo RNA birangiye, bigomba guhindurwa muri cDNA kubushakashatsi bwakurikiyeho, bityo intambwe yo guhinduka ni ngombwa.Guhindura inyandiko-mvugo bizatangizwa muguhitamo revers transcriptase na primer:

Guhindura inyandiko-mvugo ihitamo:

Inyandiko zisanzwe zinyuranye zirimo AMV RTase na MMLV RTase.RNase H ya AMV RTase ifite ibikorwa bikomeye, uburebure bwa synthesis ngufi, ingano ya synthesis nkeya hamwe nubushyuhe bwiza bwumuriro (42 ~ 55 ℃).Igikorwa cya RNase H cya MMLV RTase gifite intege nke, uburebure bwa synthesis ni ndende, ingano ya synthesis ni myinshi, kandi ubushyuhe bwumuriro ni bubi (37 ~ 42 ℃).

Kuberako enzyme ya RNase H ifite umurimo wo gutesha agaciro inyandikorugero ya RNA, MMLV hamwe nigikorwa cya RNase H idakwiye guhitamo neza mugihe cyo kwandukura, hanyuma nyuma yubwubatsi bwa geneti, nyuma yubushyuhe bwa MMLV bugeze kumurongo mwiza.Gufata ForegeneForeasy Reverse Transcriptase (M-MLV yo kwandukura) nk'urugero, ni inyandiko mvugo isubiza inyuma igaragara muri E. coli yakozwe na bagiteri ikoresheje tekinoroji ya genoside.Ni polymerase ya ADN ya recombinant ihuza umurongo wa ADN yuzuzanya kuva RNA, ADN, cyangwa RNA: Hybrid ya ADN.Ntabwo ifite ibikorwa bya RNase H, ituze rikomeye, isano ikomeye ya RNA, hamwe no kumva neza.

 

Foreasy Reverse Transcriptase (M-MLV yo kwandukura)

Guhitamo primer:

Mubisanzwe RT primers iri mubyiciro bitatu: oligo dT, primers idasanzwe, na gene yihariye.Hitamo primers ikwiye gukoreshwa ukurikije ibisabwa bitandukanye byubushakashatsi.

1. Niba inyandikorugero ikomoka kuri eukaryotic kandi cDNA yatinze ikoreshwa muburyo busanzwe bwa PCR, Oligo (dT) birasabwa;Niba igeragezwa ryakurikiyeho rikoreshwa gusa kuri qPCR, Oligo (dT) irasabwa kuvangwa na primers zidasanzwe kugirango tunoze imikorere ya transcript.

2. Niba inyandikorugero ikomoka kuri prokaryotes, Primers Primers cyangwa gene primers igomba gutoranywa kugirango ihindurwe.

Ⅲ.QPCR

Umubare wa Fluorescence urasobanurwa cyane cyane muguhitamo uburyo bwo kubara, amahame yo gushushanya primer, guhitamo ROX, sisitemu ya reaction hamwe nuburyo ibintu byifashe, nibindi.

Guhitamo uburyo bwo kubara:

Uburyo butandukanye bugabanijwe muburyo bugereranije nuburyo bwuzuye bwo kubara.Umubare ugereranije urashobora gukoreshwa kugirango umenye ingaruka zuburyo bumwe na bumwe bwo kuvura ku mvugo ya gene, kumenya itandukaniro ryimvugo ya gene mubihe bitandukanye no kugereranya itandukaniro ryimvugo ya gene mubice bitandukanye.Umubare wuzuye urashobora kumenya ingano ya acide nucleic muri virusi nibindi.Mugihe dukora ubushakashatsi, tugomba guhitamo uburyo bukwiye bwo kubara dukurikije ubushakashatsi bwacu bwite.

Amahame yo gushushanya yibanze:

Igishushanyo cya primer kuri qPCR gifitanye isano itaziguye no kongera imbaraga hamwe nibicuruzwa byihariye.Kubwibyo, gushushanya neza primers nintambwe yambere yo gutsinda qPCR.Mugushushanya primer, amahame akurikira agomba kwitabwaho mugihe yujuje ihame ryibishushanyo mbonera bisanzwe:

1. Uburebure bwigice cyagenewe kugenzurwa hagati ya 100 na 300 bp;

2. Igishushanyo cya Cross-exon kugirango wirinde ingaruka za ADN genomic;

3. Ibishushanyo mbonera byateganijwe bigomba kugeragezwa kugirango byongerwe imbaraga, kandi mugihe gusa amplification ikora igeze kurwego (90-110%) irashobora gukoreshwa mubushakashatsi bwuzuye;

4. Kwibanda kwa primer mubisanzwe bitezimbere hagati ya 0.1uM na 1.0uM.

GuhitamoINGINGO:

Muburyo bwo kugereranya ibintu, ROX irashobora guhindura itandukaniro ryinzira nziza, ikosa rya pipeti cyangwa itandukaniro ryijwi ryatewe no guhumeka hamwe na kondegene imwe, bikongera ibisubizo byibisubizo.Ariko, twakagombye kumenya ko guhitamo ROX bifitanye isano nigikoresho.Niba igikoresho cya qPCR gifite imikorere yo guhita ikosora itandukaniro riri hagati yimyobo, ntabwo ikeneye kongeramo ROX;bitabaye ibyo, ikeneye kongeramo ROX ikosora.Abafatanyabikorwa bato mu kugura reagent bagomba kuba bakurikije igikoresho cyakoreshejwe muguhitamo ROX ikwiye, irinde amakosa nyuma.

Gutegura sisitemu yo kwitwara:

Umubare wa reaction ya 20ul na 50ul urakunzwe.Ibibazo bikurikira bigomba kwitabwaho mugihe sisitemu yashyizweho:

1. Sisitemu ya reaction igomba gutegurwa no guhumeka mumashanyarazi ya ultra-isuku, ddH nshya₂O ikoreshwa kuri buri igeragezwa;

2. Buri igerageza rigomba gutegura NTC kugirango hamenyekane niba muri sisitemu harimo umwanda, kandi buri primers ikeneye gukora NTC mugihe itegura sisitemu;

3. Kugirango umenye niba hari ibisigisigi bya gDNA mubishusho bya RNA, NRT irashobora gutegurwa kuri buri sample kugirango imenye;

4. Mugihe utegura sisitemu, birasabwa gukora byibuze inshuro 3 gusubiramo tekinike imwe;

5. Iyo inyandikorugero ari cDNA, birasabwa kugabanya inshuro 5-10 kugirango ugabanye ingaruka zo kubuza sisitemu yo kwandukura ibintu ku bushakashatsi bwa qPCR.Nibyiza gushakisha urugero rwicyitegererezo ukurikije gradient, kugirango CT agaciro igabanuke hagati ya 20-30;

6. Menya umubare ukenewe wibisubizo, wongere 5-10% ukurikije umubare wibisubizo, kandi ubare umubare wiboneza;

7, sisitemu yateguwe hifashishijwe ihame ryibanze, kuvanga nyuma ya centrifugation kandi urebe ko nta bubble;

8, Mugihe gishoboka cyo guhitamo gushyigikira ibikoreshwa.

Bifitanye isano RT-qPCR Kit