Ct agaciro nigisubizo cyingenzi cyerekana uburyo bwa fluorescent quant PCR.Byakoreshejwe mukubara imvugo ya gene itandukanye cyangwa nimero ya kopi.None ni ubuhe butumwa bwa Ct bwo kugereranya fluorescence ifatwa nkibyumvikana?Nigute ushobora kwemeza urwego rwiza rwa Ct?
Agaciro Ct ni iki?
Mugihe cya qPCR yogukwirakwiza, umubare uhwanye na cycle amplification cycle (Cycle Threshold) mugihe ibimenyetso bya fluorescence yibicuruzwa byongerewe bigera kumurongo wa fluorescence.C bisobanura Cycle na T bisobanura Threshold.Muri make, Ct agaciro numubare wizenguruko uhuye nigihe inyandikorugero yambere amplification igera kumubare runaka wibicuruzwa muri qPCR.Ibyo bita "umubare runaka wibicuruzwa" bizasobanurwa nyuma.
Niki agaciro Ct ikora?
1.Isano iri hagati ya amplifisione yerekana, umubare wicyitegererezo nagaciro ka Ct
Byiza, gen muri qPCR zegeranijwe na amplification yerekanwe nyuma yumubare runaka wizunguruka.Isano iri hagati yumubare wokuzunguruka kwinshi nubunini bwibicuruzwa ni: Umubare wibicuruzwa byongerewe = umubare wambere wicyitegererezo × (1 + En) inomero yumuzingi.Nyamara, reaction ya qPCR ntabwo buri gihe iba mubihe byiza.Iyo ingano yibicuruzwa byongerewe igeze ku "bicuruzwa runaka", umubare wizunguruka muri iki gihe ni agaciro ka Ct, kandi ni mugihe cyo kwaguka.Isano iri hagati yagaciro ka Ct nubunini bwo gutangira inyandikorugero: Hariho umurongo ugaragara hagati ya Ct agaciro kicyitegererezo na logarithm yo gutangira kopi yumubare wicyitegererezo.Hejuru yicyitegererezo cyambere yibanze, ntoya Ct agaciro;hepfo intangiriro yicyitegererezo yibanze, nini ya Ct agaciro.
2.Amplification curve, fluorescence ntarengwa numubare wibicuruzwa bya PCR
Ingano yibicuruzwa bya qPCR byerekanwa muburyo butaziguye muburyo bwa signal ya fluorescent, ni ukuvuga amplification curve.Mubyiciro byambere bya PCR, amplification iri mubihe byiza, umubare wizunguruko ni muto, kwegeranya ibicuruzwa ni bito, kandi urwego rwa fluorescence ntirushobora gutandukanywa neza na fluorescence.Nyuma yibyo, fluorescence iriyongera kandi yinjira mugice cyerekana.Ingano yibicuruzwa bya PCR irashobora kumenyekana mugihe runaka mugihe reaction ya PCR iri murwego rwo kwerekana ibintu, ishobora gukoreshwa nk "ibicuruzwa runaka", kandi ibikubiyemo byambere byicyitegererezo birashobora gukurwa muribi.Kubwibyo, ibimenyetso bya fluorescence ubukana bujyanye nigicuruzwa runaka nigipimo cya fluorescence.
Mugihe cyanyuma cya PCR, amplification curve ntikigaragaza amplifisione yerekanwe, kandi yinjira kumurongo wumurongo na plateau.
3.Kwerekana agaciro ka Ct
Iyo PCR izenguruka igeze kumubare wizunguruka ya Ct agaciro, yinjiye gusa mugihe cyukuri cyo kwaguka.Muri iki gihe, ikosa rito ntabwo ryongerewe, bityo kubyara agaciro ka Ct ni byiza, ni ukuvuga, inyandikorugero imwe yongerewe ibihe bitandukanye cyangwa mu tubari dutandukanye icyarimwe.Amplification, Ct yabonetse ihoraho.
1.Ubushobozi bwo kongera imbaraga En
Gukora neza kwa PCR bivuga imikorere polymerase ihindura gene kugirango yongerwe muri amplicon.Kwiyongera kwingirakamaro iyo molekile imwe ya ADN ihinduwe molekile ebyiri za ADN ni 100%.Amplification imikorere ikunze kugaragara nka En.Kugirango byorohereze isesengura ryingingo zikurikira, ibintu bigira ingaruka kumikorere ya amplification byatangijwe muri make.
| Impamvu | ibisobanuro | Nigute ushobora guca imanza? |
| A. Inhibitori ya PCR | 1. Inyandikorugero ya ADN irimo ibintu bibuza PCR kwitwara, nka poroteyine cyangwa ibikoresho.2. CDNA nyuma yo kwandukura inyandiko ikubiyemo ibintu byinshi byerekana urugero RNA cyangwa RT reagent ibice, bishobora no kubuza PCR gukurikira. | 1. Niba hari umwanda ushobora kugenzurwa no gupima igipimo cya A260 / A280 na A260 / A230 cyangwa amashanyarazi ya RNA.2. Niba cDNA ivangwa ukurikije igipimo runaka nyuma yo kwandukura. |
| B. Igishushanyo mbonera cya primer | Primers ntabwo ifata neza | Reba primers kuri primer-dimers cyangwa umusatsi, kudahuza, kandi rimwe na rimwe uzenguruka ibishushanyo mbonera. |
| C. Igishushanyo mbonera cya PCR idakwiye | 1. Primers ntishobora gufatana neza2. Irekurwa ridahagije rya ADN polymerase 3. Ubushyuhe burebure burebure ibikorwa bya ADN polymerase byagabanutse | 1. Ubushyuhe bwa annealing buri hejuru ya TM agaciro ka primer2. Igihe cyabanjirije gutandukana ni gito cyane 3. Igihe cya buri cyiciro cyibikorwa bya reaction ni kirekire cyane |
| D. Kuvanga bidahagije bya reagent cyangwa amakosa yo gutobora | Muri sisitemu yo kubyitwaramo, kwibanda kwa PCR yibice bya reaction ni byinshi cyane cyangwa bitaringaniye, bikavamo kutongera imbaraga kwa amplification ya PCR | |
| E. Uburebure bwa Amplicon | Uburebure bwa amplicon ni ndende cyane, burenga 300bp, kandi amplification ikora ni mike | Reba neza ko uburebure bwa amplicon buri hagati ya 80-300bp |
| F. Ingaruka za reagent ya qPCR | Ubwinshi bwa ADN polymerase muri reagent ni buke cyangwa kwibumbira hamwe kwa ion muri buffer ntabwo ari byiza, bigatuma ibikorwa bya enzyme ya Taq bitagera kuri byinshi | Kugena imikorere ya amplification ikora kumurongo usanzwe |
2.Urutonde rwa Ct
Indangagaciro za Ct ziri hagati ya 15-35.Niba agaciro ka Ct kari munsi ya 15, bifatwa ko amplification iri murwego rwibanze kandi fluorescence ntarengwa itaragera.Byiza, hariho umurongo ugaragara hagati ya Ct agaciro na logarithm yumubare wambere wimibare yicyitegererezo, ni ukuvuga, umurongo usanzwe.Binyuze kumurongo usanzwe, mugihe amplification ikora ari 100%, agaciro kabaruwe Ct yo kugereranya numero imwe ya kopi ya gene igera kuri 35. Niba irenze 35, nimero yambere ya kopi yicyitegererezo iri munsi ya 1, ishobora gufatwa nkibidafite ishingiro.
Kuri gen zitandukanye Ct zingana, kubera itandukaniro ryimibare ya kopi ya gene hamwe nubushobozi bwa amplification muburyo bwambere bwicyitegererezo, birakenewe gukora umurongo usanzwe wa gene no kubara umurongo ugaragaza gene.
3.Guhindura ibintu bya Ct agaciro
Uhereye ku isano iri hagati yumubare wikwirakwizwa ryinshi nubunini bwibicuruzwa: ingano yibicuruzwa byongerewe = ingano yicyitegererezo cyambere × (1 + En) inomero yumuzingi, birashobora kugaragara ko mubihe byiza, ingano yicyitegererezo cyambere na En bizagira ingaruka mbi kuri Ct agaciro.Itandukaniro mubyitegererezo byubwiza cyangwa amplification ikora bizatera agaciro Ct kuba nini cyane cyangwa nto cyane.
4.Ct agaciro nini cyane cyangwa nto cyane
Igihe cyo kohereza: Gashyantare-22-2023
