Ibikoresho byo gutangira: RNA

Quantitative reverse transcription PCR (RT-qPCR) nuburyo bwubushakashatsi bwakoreshejwe mubushakashatsi bwa PCR ukoresheje RNA nkibikoresho byo gutangira.Muri ubu buryo, RNA yose cyangwa intumwa RNA (mRNA) yabanje kwandikwa muri ADN yuzuzanya (cDNA) na transcript transcriptase.Ibikurikira, reaction ya qPCR yakozwe hakoreshejwe cDNA nkicyitegererezo.RT-qPCR yakoreshejwe muburyo butandukanye bwa biyolojiya y’ibinyabuzima, harimo gusesengura imiterere ya gene, kwemeza RNA kwivanga, kwemeza microarray, gutahura indwara ya virusi, gupima geneti, n’ubushakashatsi bw’indwara.

Intambwe imwe nuburyo bubiri kuri RT-qPCR

RT-qPCR irashobora kugerwaho nintambwe imwe cyangwa intambwe ebyiri.Intambwe imwe RT-qPCR ikomatanya kwandukura no guhinduranya PCR, bigatuma transcriptase na reaction ya ADN polymerase kugirango irangize reaction mumiyoboro imwe mubihe bimwe bya buffer.Intambwe imwe RT-qPCR isaba gusa gukoresha primaire yihariye.Mu ntambwe ebyiri RT-qPCR, transcription transcription hamwe na PCR amplification ikorerwa mumiyoboro ibiri, ukoresheje buffers zitandukanye zitezimbere, imiterere yimikorere, hamwe nuburyo bwo gushushanya primer.

Ibicuruzwa bifitanye isano:

RT-qPCR Byoroshyeᵀᴹ (Intambwe imwe) -SYBR Icyatsi I.

RT-qPCR Byoroshyeᵀᴹ (Intambwe imwe) -Taqman

RT Byoroshyeᵀᴹ I Master Premix Kuri Cyambere-Cyambere CDNA Synthesis

Igihe nyacyo PCR Byoroshyeᵀᴹ-SYBR Icyatsi I Kit

Igihe nyacyo PCR Byoroshyeᵀᴹ-Taqman

Guhitamo RNA yose hamwe na mRNA

Mugihe utegura igeragezwa rya RT-qPCR, ni ngombwa guhitamo niba wakoresha RNA yose cyangwa mRNA isukuye nkicyitegererezo cyo kwandukura.Nubwo mRNA ishobora gutanga sensibilité yo hejuru gato, RNA yose iracyakoreshwa kenshi.Impamvu yabyo nuko RNA yose ifite inyungu zingenzi nkibikoresho byo gutangira kuruta mRNA.Ubwa mbere, inzira isaba intambwe nke zo kwezwa, zitanga uburyo bwiza bwo kugarura inyandikorugero hamwe nibisanzwe mubisubizo kugirango utangire umubare wimibare.Icya kabiri, irinda intambwe yo gukungahaza mRNA, ishobora kwirinda ibishoboka ibisubizo byoroheje bitewe no gukira kwa mRNA zitandukanye.Muri rusange, kubera ko mubisabwa byinshi ugereranije ingano ya gen igenewe ni ngombwa kuruta ibyiyumvo byuzuye byo gutahura, RNA yose irakwiriye mubihe byinshi.

Subiza inyandiko mvugo

Muburyo bwintambwe ebyiri, uburyo butatu butandukanye burashobora gukoreshwa mugutangiza cDNA reaction: oligo (dT) primers, primers random, cyangwa primaire yihariye.Mubisanzwe, oligo (dT) primers na primers zidasanzwe zikoreshwa mukomatanya.Izi primers zifatanije nicyitegererezo mRNA umurongo kandi zitanga inyandiko zinyuranye hamwe nintangiriro yo guhuza.

Hindura inyandiko mvugo

Reverse transcriptase ni enzyme ikoresha RNA muguhuza ADN.Inyandiko zimwe zinyuranye zifite ibikorwa bya RNase kandi zishobora gutesha agaciro imirongo ya RNA mumigozi ya RNA-ADN nyuma yo kwandukura.Niba idafite ibikorwa byimikorere ya RNase, RNaseH irashobora kongerwaho kugirango qPCR ikore neza.Imisemburo ikunze gukoreshwa harimo virusi ya Moloney murine leukemia reverse transcriptase na avian myeloblastoma virusi reverse transcriptase.Kuri RT-qPCR, nibyiza guhitamo inyandiko-mvugo ihindagurika hamwe nubushyuhe bwo hejuru cyane, kugirango synthesis ya cDNA ikorwe mubushyuhe bwo hejuru, byemeze neza ko RNAs yandikirwa neza kandi ifite urwego rwisumbuye rwisumbuye, mugihe ikomeza ibikorwa byayo byose mubisubizo, bikavamo umusaruro mwinshi wa cDNA.

Ibicuruzwa bifitanye isano:

Foreasy M-MLV Guhindura Inyandiko

RNase H ibikorwa bya transcript transcriptase

RNaseH ishoboye gutesha umurongo imirongo ya RNA ikomoka kuri RNA-ADN duplexes, itanga synthesis nziza ya ADN ebyiri.Ariko, mugihe ukoresheje mRNA ndende nkicyitegererezo, RNA irashobora guteshwa agaciro imburagihe, bikavamo cDNA yaciwe.Kubwibyo, akenshi ni byiza kugabanya ibikorwa bya RNaseH mugihe cDNA ikoronijwe niba hakenewe synthesis ya transcript ndende.Ibinyuranye, guhinduranya inyandiko hamwe nibikorwa bya RNase H akenshi bigira akamaro kubikorwa bya qPCR kuko byongera gushonga kwa duplexes ya RNA-ADN mugihe cyambere cya PCR.

Igishushanyo mbonera

PCR yibanze ikoreshwa kuri intambwe ya qPCR muri RT-qPCR igomba kuba yarateguwe kugirango ihuze exon-exon, aho primer amplification ishobora kuzenguruka imbibi za exon-intron.Kubera ko intangangore zirimo ADN zikurikirana zitaguwe neza, iki gishushanyo kigabanya ibyago byibyiza bibi byongerewe kwanduza ADN genomic.

Niba primers idashobora gutegurwa gutandukanya exons cyangwa imbibi za exon-exon, birashobora kuba ngombwa kuvura ingero za RNA hamwe na DNase ya RNase idafite DNase I cyangwa dsDNase kugirango ikureho ADN yanduye.

Igenzura rya RT-qPCR

Igenzura ridahinduka (-RT igenzura) rigomba gushyirwa mubigeragezo byose RT-qPCR kugirango hamenyekane umwanda wa ADN (nka ADN genomic cyangwa ibicuruzwa bya PCR bivuye mubitekerezo byabanje).Igenzura ririmo ibice byose byerekana reaction usibye guhinduranya inyandiko.Kubera ko guhinduranya inyandiko bitabaho hamwe nubu bugenzuzi, niba PCR yongerewe imbaraga, kwanduza ADN birashoboka cyane.