Kwinkqubo yokuqhuba izifundo zofuzo, sihlala sidibana neesampulu ezingonelanga ze-RNA, umzekelo, ukufunda amathumba omlomo we-anatomical, nkqu iisampulu zeseli enye, kunye neesampulu zokuguqulwa kofuzo oluthile olubhalwe kumanqanaba aphantsi kakhulu kwiiseli zabantu.Ewe kunjalo, kuvavanyo lwe-COVID-19, ukuba ii-swabs azikho kwindawo efanelekileyo okanye amaxesha anganelanga ngexesha lesampulu, ubungakanani besampulu buya kuba sezantsi kakhulu, yiyo loo nto iKhomishini yezeMpilo kunye noCwangciso lweNtsapho yaphuma kwiintsuku ezimbini ezidlulileyo kwaye. luphumelele uvavanyo, kwaye ukuba isampulu ye-nucleic acid ayizange ithathe iisampuli ezintandathu, ungayixela.
Uvakalelo lwe-reagent lubalulekile kuba sinale ngxaki okanye loo ngxaki, ngoko singenza ntoni ukuphucula uvakalelo lwe-RT-PCR?
Ngaphambi kokuba sithethe ngezicombululo ezinokubakho, makhe sikhankanye iingxaki ezimbini ezinkulu kule meko sisandul’ ukuyikhankanya.
Okokuqala, sinexhala malunga nokulahleka kwe-RNA xa sineeseli ezimbalwa kwisampulu yethu.Ukuba ukwahlukana kwendabuko kunye neendlela zokucoca zisetyenzisiweyo, njengendlela yekholomu okanye indlela ye-nucleic acid precipitation, kukho ithuba eliphezulu lokuba iisampuli ezimbalwa ziya kulahleka.Esinye isisombululo kukongeza i-molecule ephetheyo, efana ne-tRNA, kodwa nangona kunjalo, akukho siqinisekiso sokuba ilinge lethu lokubuyisela lilungile.
Ke yeyiphi indlela engcono?Inketho efanelekileyo yeeseli ezikhuliswe okanye iisampuli ze-microanatomical kukusebenzisa i-lysis ngqo.
Umbono kukwahlula iiseli imizuzu emi-5, ukukhulula i-RNA kwisisombululo, emva koko uyeke ukuphendula imizuzu emi-2, emva koko wongeze i-lysate ngokuthe ngqo kwi-reverse transcription reaction ukuze kungabikho i-RNA elahlekileyo, kwaye ekugqibeleni ubeke i-cDNA ngqo. kwimpendulo yexesha lokwenyani.
Kodwa kuthekani ukuba, ngenxa yesiqalo esilinganiselweyo okanye isixa esincinci sokubonakaliswa kofuzo, sinokuphinda sisebenzise yonke i-RNA kwaye singanikezeli ngeetemplates ezaneleyo zokufumana isiginali elungileyo yexesha lokwenyani?
Kule meko, inyathelo lokukhulisa kwangaphambili linokuba luncedo kakhulu.
Oku kulandelayo sisikimu sokwandisa ubuntununtunu emva kokukhutshelwa umva.Phambi kokuba siqale, kufuneka sibuze ezantsi komlambo ukuba zeziphi iithagethi esinomdla kuzo, ukuze siyile iiprimer ezithile zezi thagethi zokwandiswa kwangaphambili.
Oku kunokufezekiswa ngokudala i-primer edibeneyo ukuya kuthi ga kwi-100 pair of primers kunye nomjikelezo wokuphendula we-10 ukuya kwi-14 amaxesha.Ke ngoko, uMxube oyi-Master ngokukodwa owenzelwe le mfuno uyafuneka ukuze kwandiswe kwangaphambili i-cDNA efunyenweyo.
Isizathu sokumisela inani lemijikelo phakathi kwe-10 kunye ne-14 kukuba eli nani lilinganiselwe lemijikelo liqinisekisa ukungahambi kakuhle phakathi kweethagethi ezahlukeneyo, okubaluleke kakhulu kubaphandi abafuna ulwazi lwe-molecular quantitative.
Emva kokwandiswa kwangaphambili, sinokufumana inani elikhulu le-cDNA, ukwenzela ukuba uvakalelo lokubona kwi-back-end luphuculwe kakhulu, kwaye sinokuyinciphisa isampuli kwaye senze iimpendulo ze-PCR ezininzi zexesha lokwenene ukuphelisa iimpazamo ezinokwenzeka.
Ixesha lokuposa: Apr-11-2023