Tabbatar da aikin firamare da bincike a farkon matakin PCR reagents da kuma ƙayyade mafi dacewa halayen halayen sune abubuwan da ake buƙata don tabbatar da ingantaccen ci gaba na gwaje-gwaje na yau da kullun.
Don haka ta yaya muke buƙatar tabbatar da binciken farko a matakin farko?
Babban alamun sune tushen tushe, lanƙwan haɓakawa, ƙimar ct, haɓaka haɓakawa, ganowar samfurin ƙarancin hankali, CV, da sauransu.
Baseline
Tushen shine layin kwance a cikin lanƙwan haɓakawa na PCR.A cikin ƴan zagayowar farko na amsawar haɓakawa na PCR, siginar kyalli ba ya canzawa da yawa kuma yana samar da madaidaiciyar layi.Wannan madaidaiciyar layi shine tushe.
A lokacin da ake yin gwajin bincike na farko na PCR, kula da ko matakin matakin matakin.Tsaftataccen ƙaddamarwar binciken farko zai shafi tushen tushe, kamar haifar da haɓakar tushe ko faɗuwa.Tushen kuma alama ce mai saurin fahimta.
Ƙwaƙwalwar Ƙarawa
Wani alama mai hankali shine sifar ƙararrawa.Zai fi kyau a sami lanƙwan S-dimbin yawa don guje wa haɓakawa na biyu ko wasu lalurar haɓakawa mara kyau.
Ct Darajar
Adadin zagayowar da ke daidai da madaidaicin juzu'i daga tushe zuwa girma mai girma shine ƙimar Ct.
Don samfurin guda ɗaya, nau'ikan bincike daban-daban suna haifar da nau'ikan haɓakawa daban-daban, kuma ƙimar haɓakar haɓakawa da matakin tsangwama za ta shafi madaidaicin ƙimar Ct.A ka'idar, ƙaramin ƙimar Ct na binciken farko da muka zaɓa, mafi kyau.
Ingantaccen Ingantawa
Ɗaya daga cikin ingantattun hanyoyin dogaro da kwanciyar hankali don kimanta ingancin haɓakawa na PCR shine madaidaicin lanƙwasa, wanda masu bincike kuma suka san shi sosai.Hanyar ta ƙunshi yin jerin samfurori don sarrafa adadin dangi na samfuran manufa.Wadannan samfurori yawanci ana yin su ta hanyar dilutions na abubuwan da aka tattara na hannun jari, mafi yawan amfani da su shine dilution mai ninki 10.Yin amfani da jerin samfuran diluted, ta yin amfani da daidaitaccen shirin qPCR don haɓakawa don samun ƙimar Cq, kuma a ƙarshe zana madaidaicin madaidaicin daidai gwargwado bisa ga tattarawar kowane samfurin da ƙimar Cq daidai don samun madaidaicin layin Cq = -klgX0 + b, da haɓakar haɓakawa E = 10 (-1 / k) -1.Lokacin amfani da qPCR don ƙididdige ƙididdiga, ana buƙatar haɓakar haɓakawa don kasancewa cikin kewayon 90% -110% (3.6> k> 3.1).
Gano Samfuran Ƙarƙashin Tattaunawa
Lokacin da ƙaddamarwar samfurin ya yi ƙasa, ƙimar gano nau'ikan bincike daban-daban sun bambanta.Mun zaɓi samfuran ƙananan ƙira 20 don yin kwafi, kuma tsarin bincike-bincike tare da mafi girman ƙimar ganowa shine mafi kyau.
Coefficient of Bambanci (CV)
Za'a iya gano samfuran kwafi guda 10 tare da bincike na farko daban-daban bisa ga ma'aunin layin reagent don gano haɓaka haɓakar acid nucleic.
Yawan Reagents:
Daidaito
Daidaita a cikin tsari guda ya kamata ya hadu: ƙimar bambancin (CV,%) na ƙimar logarithmic na ƙaddamarwar gwajin shine ≤5%.Lokacin da ƙaddamarwar samfurin ya yi ƙasa, ƙididdiga na bambancin (CV,%) na logarithm na ƙaddamarwar ganowa shine ≤10%
Reagents masu inganci:
Daidaito
Daidaito a cikin rukuni guda ya kamata ya hadu:
(1) Ƙimar bambancin ƙimar Ct (CV,%) ≤5%
Ana gwada samfurin iri ɗaya a layi daya don sau 10, kuma sakamakon gwajin yakamata ya kasance daidai
Lokacin aikawa: Satumba 18-2021
