Momwe mungasinthire kukhudzika kwa RT-PCR kachitidwe ka RNA kuzindikira

Pochita maphunziro a majini, nthawi zambiri timakumana ndi zitsanzo za RNA zosakwanira, mwachitsanzo, pophunzira zotupa zazing'ono zapakamwa, ngakhale zitsanzo za cell imodzi, ndi zitsanzo za masinthidwe enieni a jini omwe amalembedwa pamilingo yotsika kwambiri m'maselo amunthu.Zachidziwikire, pakuyezetsa kwa COVID-19, ngati ma swabs sali pamalo oyenera kapena osakwanira nthawi yoyeserera, kukula kwa zitsanzo kumakhala kotsika kwambiri, ndichifukwa chake Commission of Health and Family Planning idatuluka masiku awiri apitawa. anapambana mayeso, ndipo ngati nucleic acid sampler sanatenge zitsanzo zisanu ndi chimodzi, mukhoza kunena.

Kukhudzika kwa reagent ndikofunikira chifukwa tili ndi vuto ili kapena vutolo, ndiye tingachite chiyani kuti tipititse patsogolo chidwi cha RT-PCR?

Tisanakambirane njira zothetsera vutoli, tiyeni tikambirane mavuto awiri aakulu amene angabwere chifukwa cha zimene tatchulazi.

Choyamba, timadandaula za kutayika kwa RNA pamene tili ndi maselo ochepa chabe mu zitsanzo zathu.Ngati njira zachikhalidwe zolekanitsa ndi kuyeretsa zikugwiritsidwa ntchito, monga njira yazanja kapena njira ya mpweya wa nucleic acid, pali kuthekera kwakukulu kuti zitsanzo zochepa zitha kutayika.Njira imodzi ndiyo kuwonjezera molekyulu chonyamulira, monga tRNA, koma ngakhale pamenepo, palibe chitsimikizo kuti kuyesa kwathu kuchira kuli bwino.

Ndiye njira yabwino ndi iti?Njira yabwino yama cell otukuka kapena zitsanzo za microanatomical ndikugwiritsa ntchito lysis mwachindunji.

Lingaliro ndi kugawa maselo kwa mphindi 5, kumasula RNA mu yankho, ndiye kusiya zomwe kwa mphindi 2, kenaka yikani lysate mwachindunji ku reverse transcription reaction kuti palibe RNA yotayika, ndipo potsiriza ikani zotsatira za cDNA mwachindunji. muzochitika zenizeni.

Koma bwanji ngati, chifukwa cha poyambira pang'ono kapena kachulukidwe kakang'ono ka jini ya chandamale, tingathe kubwezeretsanso RNA yonse koma osapereka ma tempuleti okwanira kuti tipeze chizindikiro chabwino cha nthawi yeniyeni?

Pankhaniyi, sitepe yokulitsa ingakhale yothandiza kwambiri.

Zotsatirazi ndi chiwembu chowonjezera chidwi pambuyo polemba mosintha.Tisanayambe, tiyenera kufunsa kunsi kwa mitsinje kuti ndi mipata iti yomwe tikufuna, kuti tipange zoyambira zazomwezi kuti tizikulitsa.

Izi zitha kuchitika popanga choyambira chosakanikirana chokhala ndi mapeyala 100 a zoyambira ndikuzungulira ka 10 mpaka 14.Chifukwa chake, Master Mix yopangidwira makamaka izi imafunikira kuti mukweze cDNA yopezedwa.

Chifukwa choyika chiwerengero cha mizere pakati pa 10 ndi 14 ndikuti chiwerengero chochepa cha maulendowa chimatsimikizira kuti palibe pakati pa zolinga zosiyanasiyana, zomwe ndizofunikira kwambiri kwa ofufuza omwe amafunikira chidziwitso cha ma molekyulu.

Pambuyo pakukulitsa chisanadze, titha kupeza kuchuluka kwa cDNA, kotero kuti chidziwitso chodziwikiratu chakumbuyo chakumbuyo chimakhala bwino kwambiri, ndipo titha kutsitsanso chitsanzocho ndikuchita zingapo zenizeni zenizeni za PCR kuti tithetse zolakwika zomwe zingachitike mwachisawawa.