RT-qPCR nubushakashatsi bwibanze bwibinyabuzima bya molekuline, kandi buri wese agomba kubimenyera.Harimo cyane cyane intambwe eshatu: Gukuramo RNA, guhinduranya inyandiko muri cDNA, hamwe na PCR yibihe nyabyo PCR.Ntabwo bifasha, bigenda bite?Birashoboka ko hari ikibazoigeragezwa ryo kwandukura!Nubwo bisa nkaho igeragezwa ryinyandiko ikeneye gusa kongeramo RNA, dNTP, primers, naInyandikomvugokuri centrifuge tube hanyuma ukavanga neza, ariko mubikorwa nyirizina, haracyari byinshi birambuye bigomba kwitabwaho.Reka tubyige!

Nigute dushobora gusuzuma ubuziranenge bwa RNA?
Kubona cDNA, ubwiza bwa RNA burakomeye!Ubwiza bwa RNA bushobora kugaragara cyane cyane mubice bibiri:
(1) Ubunyangamugayo bwa RNA:Ubunyangamugayo bwa RNA bushobora kugenzurwa na agarose gel electrophorei. Dufashe urugero rwa eukaryote, RNA yuzuye ifite imirongo itatu isobanutse, uburemere bwa molekile kuva nini kugeza nto ni 28S, 18S, na 5S, na 28S ikubye kabiri 18S;niba imirongo itatu ishobora kugaragara, ariko ubwoko bwitsinda bwaravanze cyangwa Diffusion bivuze ko RNA yangiritse igice.Muri iki gihe, nyamuneka kora reaction ya reaction ya reaction hanyuma wongere inyandikorugero yinjiza muburyo bukwiye;niba gusa itsinda rifite uburemere buke bwa molekile cyangwa ntagishobora kuboneka, RNA yarangiritse rwose kandi igomba kongera gukururwa.Agilent 2100 yerekana ubunyangamugayo bwa RNA hamwe nigishushanyo mbonera nagaciro ka RIN.Niba aside nucleic idahwitse, ibyingenzi bya electropherogramme birasa;niba aside nucleic yangiritse cyane, ibyingenzi ntibingana kandi impinga nyinshi zo kwangirika zigaragara;agaciro ka RIN kagaragaza ubunyangamugayo bwa RNA, murwego rwa 0-10, uko agaciro kangana, nibyiza bya RNA.Nibyiza, urwego rwo hejuru rwuzuye.
(2) Ubuziranenge bwa RNA:Umubare wa OD260 / 280 urashobora gutahurwa na UV spectrophotometry.Niba igipimo cya OD260 / 280 kiri hagati ya 1.9 na 2.1, ubuziranenge nibyiza cyane.
ADN isigaye ya ADN irashobora kuganisha ku bisubizo bitari byo
Iyo RNA ikuweho, RNA tubona irashobora kuvangwa na ADN genomic (gDNA) itarasukurwa.Kubwibyo, cDNA nyuma yo kwandukura inyandiko nayo izavangwagDNA.Mugihe cyo kumanukaQPCRreaction,cDNAna gDNA irashobora kongerwaho icyarimwe, bikavamo agaciro gake CT, ibisubizo rero birashobora kubogama.
None dukwiye gukora iki muri ibi bihe?Foregeneatanga igitekerezo:
(1) Kora isuku ya genome kuri RNA ihinduwe, ishobora gukurwaho no gukuramo inkingi mugihe cyo gukuramo RNA;
(2) Koresha RNA yakuwe hamwe na DNaseI , ariko ukarangiza hamwe na EDTA;
ya reaction ya reagenthamwe na genome yo gukuraho modules;

Nigute ushobora guhitamo primers kugirango uhindure transcript?
Guhindura inyandiko ya primers nayo igira ingaruka kubisubizo bya reaction ya reaction.Urashobora guhitamo primers zidasanzwe, Oligo dT cyangwa gene yihariye ya primers kugirango uhindure transcript ukurikije ibihe byihariye byubushakashatsi:
(1) Inyandiko-mvugo yihariye: gene yihariye ya primers irasabwa;
(2) Inyandiko mvugo ndende: Oligo dT / gene yihariye yibanze irasabwa;
(3) Ibice by'imbere by'inyandiko ndende: gene yihariye yibanze / primers idasanzwe / primers idasanzwe + Oligo dT.Niba igeragezwa rya qPCR ryakurikiyeho, Oligo dT ntishobora gukoreshwa wenyine, kuko gukoresha Oligo dT yonyine bishobora gutera 3 ′ kurangiza kubogama, biganisha kubisubizo bya qPCR bidahwitse;
(4) miRNA: Stem-loop primers cyangwa tailing primers irashobora gukoreshwa.

Ni kangahe ibicuruzwa biva mu mahanga cDNA bigomba guhindurwa kugirango bigererwe?
Nyuma yo kubona cDNA yibicuruzwa byandikirwa inyuma, ni kangahe cDNA igomba kuvangwa kubushakashatsi bwa qPCR ni ngombwa cyane.Niba intumbero ya cDNA iri hejuru cyane cyangwa iri hasi cyane, imikorere ya amplification irashobora kugira ingaruka.Ibipimo bya cDNA birashobora gupimwa, kandi bigomba gukorwa gute?
.Muri iki gihe, inshuti zimwe zizavuga, noneho nzapima kwibanda nyuma yo kwezwa;hano, Foregene arashaka kwibutsa ko cDNA idasabwa kwezwa, kuko uburebure bwa cDNA bwabonetse kubisubiramo buratandukanye, kandi cDNA ngufi izabura mugusukura.
(2) Noneho gukora iki?Mbere yikigereranyo cya qPCR, igipimo cya dilution ya cDNA gishobora kugenwa binyuze mubushakashatsi.Kurugero: koresha igisubizo cya cDNA, inshuro 10, hamwe ninshuro 100 nkicyitegererezo cyibigeragezo bya qPCR, hanyuma uhitemo ibintu byoguhindura hamwe nagaciro ka CT murwego rwa 18-28.

Nigute miRNAs igomba guhindurwa?
miRNA ni molekile ntoya ya RNA ifite ubunini bungana na 22 nt idakora code ya proteine.Kubera uburebure bwayo bugufi, uburyo busanzwe bwa qPCR buragoye kubigereranya muburyo butaziguye, kubwibyo akenshi birakenewe kwagura miRNA;uburyo bukoreshwa muburyo bwo guhinduranya inyandiko ya miRNA harimo uburyo bwa stem-loop nuburyo bwo kudoda.
Uburyo bwa stem-loop nuburyo bwo kwagura miRNA wongeyeho stem-loop primers.Ubu buryo bwo gutahura bufite ibyiyumvo byihariye kandi byihariye, ariko ibyinjira byinjira ni bike.Inyandiko imwe ihindagurika irashobora kumenya gusa miRNA imwe hamwe nimbere;uburyo bwo kongeramo umurizo bugizwe na bibiri Byarangiye nigikorwa gihuriweho na enzymes ebyiri, arizo PolyA polymerase na transcript transcriptase.PolyA polymerase ishinzwe kongeramo umurizo wa PolyA kuri miRNA kugirango yongere uburebure bwayo, kandi transcriptase ikora reaction ya reaction.Ubu buryo bufite uburyo bwinshi bwo gutahura kandi bushobora kumenya miRNA nyinshi hamwe n’imbere mu nyandiko imwe ihinduranya, ariko ibyiyumvo byihariye kandi byihariye biri hasi muburyo bwa stem-loop.