Enqubweni yokwenza izifundo zofuzo, sivamise ukuhlangana namasampuli e-RNA anganele, isibonelo, okutadisha izimila zomlomo ze-anatomical, ngisho namasampula eseli elilodwa, namasampuli okuguqulwa kwezakhi zofuzo ezithile ezilotshwa kumazinga aphansi kakhulu kumaseli omuntu.Kunjalo, ekuhlolweni kwe-COVID-19, uma ama-swabs engekho endaweni efanele noma izikhathi ezinganele ngesikhathi sokusampula, usayizi wesampula uzobe uphansi kakhulu, yingakho iKhomishini Yezempilo Nokuhlela Komndeni yaphuma ezinsukwini ezimbili ezedlule futhi. kuphumelele ukuhlolwa, futhi uma isampula ye-nucleic acid ingathathanga amasampula ayisithupha, ungabika.
Ukuzwela kwe-reagent kubalulekile ngoba sinale nkinga noma leyo nkinga, ngakho-ke yini esingayenza ukuze sithuthukise ukuzwela kwe-RT-PCR?
Ngaphambi kokuba sixoxe ngezixazululo ezingase zibe khona, ake sikhulume ngezinkinga ezimbili ezinkulu ngesimo esisanda kusisho.
Okokuqala nje, sikhathazeka ngokulahleka kwe-RNA uma sinezibalo ezimbalwa zamaseli kusampula yethu.Uma kusetshenziswa izindlela ezijwayelekile zokuhlukanisa nezindlela zokuhlanza, ezifana nendlela yekholomu noma indlela ye-nucleic acid precipitation, kunethuba elikhulu lokuthi amasampula ambalwa azolahleka.Isixazululo esisodwa ukungeza i-molecule yenkampani yenethiwekhi, njenge-tRNA, kodwa noma kunjalo, asikho isiqinisekiso sokuthi ukuhlolwa kwethu kokuthola KULUNGILE.
Ngakho iyiphi indlela engcono?Inketho enhle yamaseli akhulisiwe noma amasampula e-microanatomical ukusebenzisa i-lysis eqondile.
Umqondo uwukuhlukanisa amaseli imizuzu emi-5, ukhulule i-RNA esixazululweni, bese umisa ukusabela imizuzu emi-2, bese wengeza i-lysate ngokuqondile ekuphenduleni kokubhala okuphambene ukuze kungabikho i-RNA elahlekile, futhi ekugcineni ubeke i-cDNA ewumphumela ngokuqondile. ekuphenduleni kwesikhathi sangempela.
Kodwa kuthiwani uma, ngenxa yesiqalo esinomkhawulo noma inani elincane lokuvezwa kofuzo oluqondiwe, singakwazi ukugaya kabusha yonke i-RNA kodwa singanikezi izifanekiso ezanele ukuze sithole isignali yesikhathi sangempela esihle?
Kulokhu, isinyathelo sokukhulisa kusengaphambili singaba usizo kakhulu.
Okulandelayo isikimu sokwandisa ukuzwela ngemva kokuloba okuhlanekezelwe.Ngaphambi kokuthi siqale, sidinga ukubuza ezansi komfula ukuthi yiziphi izinhloso esizithandayo, ukuze sidizayine iziqalo ezithile zalezi zinhloso zokukhuliswa kwangaphambili.
Lokhu kungafezwa ngokwakha i-primer exubile enamapheya ayi-100 weziqalo kanye nomjikelezo wokusabela wezikhathi eziyi-10 kuye kweziyi-14.Ngakho-ke, i-Master Mix eyakhelwe ngokukhethekile le mfuneko iyadingeka ukuze kuthuthukiswe i-cDNA etholiwe.
Isizathu sokusetha inani lemijikelezo phakathi kwe-10 ne-14 ukuthi le nombolo elinganiselwe yemijikelezo iqinisekisa ukungahleliwe phakathi kokuhlosiwe okuhlukahlukene, okubalulekile kubacwaningi abadinga ulwazi lwe-molecular quantitative.
Ngemva kokukhulisa kusengaphambili, singathola inani elikhulu le-cDNA, ukuze ukuzwela kokuthola ekugcineni kuthuthukiswe kakhulu, futhi singase sinciphise isampula futhi senze ukusabela kwe-PCR kwesikhathi sangempela okuningi ukuze sisuse amaphutha angaba khona angahleliwe.
Isikhathi sokuthumela: Apr-11-2023