Abantu bose baravuga ihame rya qRT-PCR igerageza, igishushanyo mbonera, gusobanura ibisubizo, nibindi, ariko ndatekereza ko ngomba gusangira nawe imikorere yubushakashatsi bwa qRT-PCR.Nibito, ariko bijyanye nibisubizo.

Mbere yo gukora qRT-PCR, dukeneye gusobanukirwa neza RNA yacu nuburyo bukoreshwa.Nyuma ya byose, imbaraga zacu zigamije kubona ibisubizo, aho kwitoza gusa.Mbere rero yo gukora qRT-PCR, dukeneye kumenya ibibazo bikurikira (bimwe muribi bireba SYBR gusa).

1 Uzi neza ko RNA yawe itateshejwe agaciro?

NanoDrop 2000 irashobora kumenya gusa ubunini bwa RNA nubuziranenge, ariko ntishobora kumenya ubusugire bwa RNA.

Agaciro RNA (RNA Intesity Number) irashobora kwerekana ubusugire bwa RNA, igaragazwa na sisitemu ya Agilent 2100 Bioanalyser.

Igishushanyo mbonera cyerekana agaciro ka RIN kuburugero rwa RNA zitandukanye (eukaryote)

Nyamara, laboratoire muri rusange ntabwo zifite Agilent 2100 Bioanalyser.Mur'ibi bihe, turashobora gutahura dukoresheje gel ya fordehide, ariko ibisabwa kumubare wuzuye wa RNA ni mwinshi, kuburyo bwihuse rero ni ugukoresha gel electrophorei isanzwe.Irasabwa kuba mubidukikije bidafite nuclease, birakenewe rero koza ikigega cya electrophoreis, icupa rya sol, gel bracket hamwe namazi ya DEPC.Agarose nayo idafite nuclease (igihe cyose ifunguye vuba), kandi Loading Buffer igomba gufungurwa bishya bishoboka, hamwe na gel 1,2%.

Menya ko gele igomba gushonga burundu, bitabaye ibyo bizatera imirongo idahwitse, nkuko bigaragara muri sample 9 mumashusho.Niba voltage ari ndende cyane cyangwa ikora igihe kinini cyane izabyara ubushyuhe kandi itume RNA yangirika, bityo voltage nigihe bigomba kugenzurwa muburyo bukwiye.Byongeye kandi, kwiruka kwa gel birashobora kandi kumenya niba muri ADN hasigaye ibisigisigi bya ADN, hanyuma ukareba niba hari umubare munini wabigenewe mu iriba.

Igishushanyo.Gel electrophoreis yerekana RNA

2 Wizeye neza kubijyanye na concentration ya cDNA yawe?

Ubunararibonye bwabavandimwe bakuru muri laboratoire nuko cDNA ya sisitemu 20 ul sisitemu yabonetse na buri inversion ivangwa 20X, mugihe bashiki bacu nyuma ya dogiteri bavanze 10X.Nkunze guterwa nikibazo.Kuberako ubwiza bwa RNA buvuzwe na buri muntu buratandukanye, urwego rwo guhinduka narwo ruratandukanye, kandi tekinoroji yo guhinduka ntishobora kuba ihamye.

Igihe cyose rero mbonye cDNA ihinduwe, nzabanza kuyinyeganyeza inshuro zigera kuri 3, hanyuma nkoreshe gene yo murugo kugirango ikore RT-PCR, umubare wizunguruka muri rusange ni inzinguzingo 25, kugirango menye icyerekezo cyihariye, hanyuma menye ibintu byanyuma.

3 Uzi neza ko primers yawe yoroshye gukoresha?

Irashobora kunyura kumurongo wa qRT-PCR, ariko ibi biracyagura amafaranga.Kuri laboratoire idafite amafaranga menshi, iyo babonye primers nyinshi, barashobora gukoresha RT-PCR isanzwe kugirango barebe niba ari itsinda rimwe bakamenya umwihariko wa primers.Niba laboratoire itabura amafaranga, umwihariko wa primers zose urashobora kumenyekana rimwe unyuze kumurongo.

4 Uzi neza ko imiterere yawe yubushakashatsi ikwiye?

SYBR igomba kurindwa urumuri rukomeye, gerageza rero uzimye urumuri rwo hejuru mugihe wongeyeho reagent ya SYBR, kandi ukeneye gusa gukoresha urumuri ruciriritse kugirango urangize.

Bika SYBR kuri 4 ° C.Mugihe ukoresheje, hinduranya witonze hejuru no hepfo kugirango uvange neza kugirango wirinde kubira ifuro, kandi ntukavunike cyane.

Bashiki bacu bamwe bato bakunda gushushanya ku kibaho cya PCR kubera gutinya kuvanga ingero, nibeshya.Kuberako ibimenyetso byawe bishoboka cyane ko bigira ingaruka ku ikusanyamakuru rya fluorescent, muri rusange ndasaba abangavu gukoresha amakaye yubushakashatsi kugirango bafashe kwibuka, nkuko bigaragara hano hepfo.

Igishushanyo.qRT-PCR icyitegererezo cyo gupakira

5 Uzi neza ko ubikora neza?

Witondere kwambara uturindantoki, kwambara uturindantoki, kwambara uturindantoki, no kuvuga ibintu by'ingenzi inshuro eshatu.

Kugirango ugabanye SYBR kumurika, njye kubwanjye nkunda kongeramo icyitegererezo mbere, nkuko bigaragara mumashusho hepfo.Ukurikije ubunararibonye, ​​kongeramo umubare muto wicyitegererezo birashoboka gutera amakosa yo gutoranya.Kubwibyo, kugirango ugabanye amakosa yatewe no kongeramo umubare muto wicyitegererezo, mubisanzwe nongeye gukuba kabiri icyitegererezo, kandi nkikuba kabiri iyo wongeyeho icyitegererezo kugirango ugabanye umubare wa H2O2 wongeyeho.

Igishushanyo.Igishushanyo mbonera cya qRT-PCR yikuramo

Noneho shiraho sisitemu ya qRT-PCR kuburyo bukurikira.

Igishushanyo.Igishushanyo mbonera cya qRT-PCR

ICYITONDERWA: Igenamiterere rigomba gukorwa ku rubura.

Nyuma yo kongeramo icyitegererezo, andika firime ifunze neza.Gerageza kudakora hejuru ya firime ya kashe ibonerana ukoresheje amaboko yawe, kora gusa uhereye kumwanya uri kumpande zombi za firime.Kuberako igikumwe gishobora no kugira ingaruka ku ikusanyamakuru rya fluorescent.Noneho koresha centrifuge kugirango centrifuge byihuse kuri 10 s kumuvuduko muke kugirango wirinde icyitegererezo kumanikwa kurukuta.

Ibicuruzwa bifitanye isano:

Akagari ka RT-qPCR kit

RT Byoroshye II