Nigute ushobora kunoza ibyiyumvo bya RT-PCR kugirango tumenye RNA

Muburyo bwo gukora ubushakashatsi bwerekeranye nubuzima, dukunze guhura ningero za RNA zidahagije, kurugero, kugirango twige ibibyimba bito byo mu kanwa, ndetse na selile imwe, hamwe nicyitegererezo cyimiterere ya gene yihariye yandukuwe kurwego rwo hasi cyane mungirangingo zabantu.Birumvikana ko ku kizamini cya COVID-19, niba swabs zitari ahantu heza cyangwa ntizihagije mugihe cyo gutoranya, ingano yicyitegererezo izaba mike cyane, niyo mpamvu komisiyo yubuzima no kuboneza urubyaro yasohotse hashize iminsi ibiri kandi yatsinze ikizamini, kandi niba nucleic aside sampler itarafashe ingero esheshatu, urashobora kubimenyesha.

Imyumvire ya reagent ni ngombwa kuko dufite iki kibazo cyangwa kiriya kibazo, none twokora iki kugirango tunoze sensibilité ya RT-PCR?

Mbere yo kuganira kubisubizo bishoboka, reka tuvuge ibibazo bibiri bikomeye hamwe nikibazo tumaze kuvuga.

Mbere ya byose, duhangayikishijwe no gutakaza RNA mugihe dufite selile nkeya gusa murugero rwacu.Niba uburyo bwa gakondo bwo gutandukana no gukora isuku bukoreshwa, nkuburyo bwinkingi cyangwa uburyo bwo kugwa kwa acide nucleic, birashoboka cyane ko ingero nke zabuze.Igisubizo kimwe nukwongeramo molekile yabatwara, nka tRNA, ariko nubwo bimeze bityo, nta garanti yerekana ko igeragezwa ryacu ryo gukira ari ryiza.

Nubuhe buryo bwiza kuruta ubundi?Ihitamo ryiza ryingirabuzimafatizo cyangwa microanatomical sample ni ugukoresha lysis itaziguye.

Igitekerezo nukugabanya selile muminota 5, kurekura RNA mugisubizo, hanyuma uhagarike reaction muminota 2, hanyuma ongeraho lysate muburyo butaziguye kugirango reaction ya reaction itagira RNA ibura, hanyuma ushireho cDNA yavuyemo. in the real-time reaction.

Ariko tuvuge iki niba, kubera aho bigarukira cyangwa umubare muto wa gene igamije, dushobora kongera gukoresha RNA yose kandi ntidutange inyandikorugero zihagije kugirango tubone ikimenyetso cyiza-gihe?

Muri iki kibazo, intambwe ibanziriza amplification irashobora kuba ingirakamaro cyane.

Ibikurikira ni gahunda yo kongera sensibilité nyuma yo kwandukura.Mbere yo gutangira, dukeneye kubaza epfo na ruguru intego dushishikajwe, kugirango dushushanye primers zihariye kuri izi ntego zo kubanziriza amplification.

Ibi birashobora kugerwaho mugukora primer ivanze hamwe na joriji 100 za primers hamwe na reaction ya inshuro 10 kugeza 14.Kubwibyo, Master Mix yateguwe kubwiki gisabwa irakenewe mbere yo kongera cDNA yabonye.

Impamvu yo gushiraho umubare wizunguruka hagati ya 10 na 14 nuko iyi mibare mike yinzinguzingo ituma habaho guhinduka hagati yintego zitandukanye, zikaba ari ingenzi kubashakashatsi bakeneye amakuru ya molekile yuzuye.

Nyuma yo kubanziriza-amplification, dushobora kubona umubare munini wa cDNA, kugirango sensibilité yo gutahura kumpera yinyuma iratera imbere cyane, ndetse dushobora no kugabanya urugero hanyuma tugakora reaction nyinshi-nyayo-PCR kugirango dukureho amakosa ashobora guterwa.