Yadda ake haɓaka hankalin RT-PCR don gano RNA

A yayin gudanar da nazarin kwayoyin halitta, sau da yawa muna cin karo da rashin isassun samfuran RNA, misali, don nazarin ƙananan ciwace-ciwacen baka, har ma da samfuran tantanin halitta guda ɗaya, da samfuran takamaiman maye gurbi waɗanda aka rubuta a ƙananan matakan a cikin ƙwayoyin ɗan adam.Tabbas, don gwajin COVID-19, idan swabs ɗin ba su kasance a wurin da ya dace ba ko kuma ba su isa lokacin yin samfur ba, girman samfurin zai yi ƙasa sosai, wanda shine dalilin da ya sa Hukumar Lafiya da Tsarin Iyali ta fito kwanaki biyu da suka gabata. wuce gwajin, kuma idan samfurin nucleic acid bai dauki samfurori shida ba, zaku iya ba da rahoto.

Hankali na reagent yana da mahimmanci saboda muna da wannan matsala ko waccan matsalar, don haka menene zamu iya yi don haɓaka hankalin RT-PCR?

Kafin mu tattauna yiwuwar mafita, bari mu ambaci manyan matsaloli guda biyu tare da yanayin da muka ambata.

Da farko, muna damuwa game da asarar RNA lokacin da muke da ƴan adadin tantanin halitta a cikin samfurin mu.Idan ana amfani da hanyoyin rarrabuwa na al'ada da tsaftacewa, kamar hanyar shafi ko hanyar hazo na acid nucleic, akwai babban yuwuwar cewa 'yan samfuran za su rasa.Ɗaya daga cikin mafita ita ce ƙara kwayoyin halitta, kamar tRNA, amma duk da haka, babu tabbacin cewa gwajin mu na murmurewa yayi kyau.

To mecece hanya mafi kyau?Kyakkyawan zaɓi don ƙwayoyin al'ada ko samfuran microanatomical shine amfani da lysis kai tsaye.

Manufar ita ce a raba sel na minti 5, a saki RNA a cikin maganin, sannan a dakatar da amsa na minti 2, sa'an nan kuma ƙara lysate kai tsaye zuwa yanayin jujjuyawar don kada RNA ta ɓace, a ƙarshe sanya cDNA da aka samu kai tsaye. a cikin ainihin-lokaci dauki.

Amma menene idan, saboda ƙayyadaddun wurin farawa ko ƙaramin adadin maganan kwayoyin halitta, za mu iya sake sarrafa duk RNA kuma har yanzu ba mu samar da isassun samfura don samun sigina mai kyau na ainihin lokacin ba?

A wannan yanayin, matakin haɓakawa na farko na iya zama da amfani sosai.

Mai zuwa wani tsari ne don ƙara hankali bayan an juyar da rubutu.Kafin farawa, muna buƙatar tambayar ƙasa ko wane maƙasudi ne muke sha'awar, ta yadda za a ƙirƙira takamaiman maƙasudai don waɗannan makasudin don haɓakawa.

Ana iya samun wannan ta hanyar ƙirƙirar haɗaɗɗen firam ɗin tare da har zuwa nau'i-nau'i 100 na al'ada da sake zagayowar amsawar sau 10 zuwa 14.Don haka, ana buƙatar Jagora Mix musamman da aka ƙera don wannan buƙatun don haɓaka cDNA da aka samu.

Dalilin saita adadin zagayowar tsakanin 10 zuwa 14 shine cewa wannan ƙayyadadden adadin kewayon yana tabbatar da bazuwar tsakanin maƙasudai daban-daban, wanda ke da mahimmanci ga masu bincike waɗanda ke buƙatar bayanan ƙwayoyin cuta masu ƙima.

Bayan pre-amplification, za mu iya samun babban adadin cDNA, sabõda haka, gane ganewa a baya-karshen da aka ƙwarai inganta, kuma za mu iya ko da tsarma samfurin da kuma yi mahara real-lokaci PCR halayen don kawar da yiwuwar bazuwar kurakurai.