Inani le-CT liwuhlobo lokwethula umphumela obaluleke kakhulu we-PCR ye-fluorescent quantitative.Isetshenziselwa ukubala umehluko wokuchazwa kofuzo noma inombolo yekhophi yofuzo.Ngakho-ke liyini inani le-Ct le-fluorescence quantification elithathwa njengelinengqondo?Ungaqinisekisa kanjani ububanzi obusebenzayo bevelu ye-CT?
Iyini i-Ct Value?
Ngesikhathi senqubo yokukhulisa i-qPCR, inombolo ehambisanayo yemijikelezo yokukhulisa (Cycle Threshold) lapho isignali ye-fluorescence yomkhiqizo okhulisiwe ifinyelela embundwini we-fluorescence omisiwe.UC umele iCycle kuthi T imele iThreshold.Kalula nje, inani le-Ct linani lemijikelezo ehambisana nalapho ukukhulisa isifanekiso sokuqala kufinyelela inani elithile lomkhiqizo ku-qPCR.Okubizwa ngokuthi “inani elithile lomkhiqizo” kuzobuye kuchazwe kamuva.
Yenzani inani le-Ct?
1.Ubudlelwano phakathi kokukhulisa i-exponential, inani lesifanekiso kanye nevelu ye-Ct
Ngokufanelekile, izakhi zofuzo ku-qPCR ziqoqwa ngokukhuliswa komchazi ngemva kwenani elithile lemijikelezo.Ubudlelwano phakathi kwenani lemijikelezo yokukhulisa kanye nenani lemikhiqizo yilokhu: Inani lomkhiqizo elikhulisiwe = inani lesifanekiso sokuqala × (1+En) inombolo yemijikelezo.Nokho, ukusabela kwe-qPCR akuhlali kusesimweni esikahle.Uma inani lomkhiqizo okhulisiwe lifinyelela "inani elithile lomkhiqizo", inani lemijikelezo ngalesi sikhathi inani le-Ct, futhi lisenkathini yokukhulisa umchazi.Ubudlelwano phakathi kwevelu ye-Ct kanye nenani lesifanekiso sokuqala: Kunobudlelwano bomugqa phakathi kwevelu ye-Ct yesifanekiso kanye ne-logarithm yenombolo yekhophi eqalayo yesifanekiso.Uma kuphakeme ukugxiliswa kwesifanekiso sokuqala, liba lincane inani le-Ct;lapho kuncipha ukugxiliswa kwesifanekiso sokuqala, inani le-Ct likhulu.
2.Ijika le-Amplification, i-fluorescence threshold kanye nenani elithile lomkhiqizo we-PCR
Inani lomkhiqizo wokukhulisa i-qPCR livezwa ngokuqondile ngendlela yesignali ye-fluorescent, okungukuthi, ijika lokukhulisa.Esigabeni sokuqala se-PCR, ukukhuliswa kungaphansi kwezimo ezifanele, inani lemijikelezo lincane, ukuqoqwa komkhiqizo kuncane, futhi izinga le-fluorescence alikwazi ukuhlukaniswa ngokucacile nesizinda se-fluorescence.Ngemva kwalokho, i-fluorescence iyanda futhi ingena esigabeni se-exponential.Inani lomkhiqizo we-PCR lingatholwa endaweni ethile lapho ukusabela kwe-PCR kusesigabeni sokuchayeka, esingasetshenziswa “njengenani elithile lomkhiqizo”, futhi okuqukethwe kokuqala kwesifanekiso kungathathwa kulokhu.Ngakho-ke, ukuqina kwesignali ye-fluorescence ehambisana nenani elithile lomkhiqizo umkhawulo we-fluorescence.
Esigabeni sakamuva se-PCR, ijika lokukhulisa alisabonisi ukukhuliswa kwe-exponential, futhi lingena esigabeni somugqa kanye nesigaba sethafa.
3.Ukukhiqizwa kabusha kwamanani e-CT
Uma umjikelezo we-PCR ufinyelela inombolo yomjikelezo yenani le-Ct, usanda kungena esikhathini sangempela sokukhulisa umchazi.Ngalesi sikhathi, iphutha elincane alizange likhuliswe, ngakho-ke ukuphindaphinda kwenani le-Ct kuhle kakhulu, okungukuthi, ithempulethi efanayo ikhuliswa ngezikhathi ezahlukene noma kumashubhu ahlukene ngesikhathi esifanayo.Ukukhulisa, inani le-Ct elitholiwe lihlala njalo.
1.Ukukhulisa kahle En
Ukusebenza kahle kokukhulisa i-PCR kubhekisela ekusebenzeni kahle i-polymerase eguqula ngayo isakhi sofuzo ukuze sikhuliswe sibe i-amplicon.Ukusebenza kahle kokukhulisa lapho i-molecule ye-DNA eyodwa iguqulwa ibe ama-molecule amabili e-DNA ingu-100%.Ukusebenza kahle kokukhulisa ngokuvamile kuvezwa njenge-En.Ukuze kube lula ukuhlaziya ama-athikili alandelayo, izici ezithinta ukusebenza kahle kokukhulisa kwethulwa kafushane.
| Izici ezinomthelela | incazelo | Indlela yokwahlulela? |
| A. PCR inhibitors | 1. I-DNA yesifanekiso iqukethe izinto ezivimbela ukusabela kwe-PCR, njengamaphrotheni noma okokuhlanza.2. I-cDNA ngemva kokuloba okuhlanekezelwe iqukethe ukugxiliswa okuphezulu kwesifanekiso se-RNA noma izingxenye ze-RT, ezingase futhi zivimbele ukusabela kwe-PCR okulandelayo. | 1. Ukuthi kukhona ukungcola kungahlulelwa ngokulinganisa isilinganiso se-A260/A280 ne-A260/A230 noma i-RNA electrophoresis.2. Ukuthi i-cDNA ihlanjululwe ngokwesilinganiso esithile ngemva kokuloba okuhlanekezelwe. |
| B. Idizayini ye-primer engafanele | Ama-primers awasebenzi kahle | Hlola ama-primer-dimers noma izikhonkwane zezinwele, ukungafani, futhi kwesinye isikhathi amadizayini aqalayo. |
| C. Umklamo wohlelo lokusabela we-PCR ongafanele | 1. Ama-primers awakwazi ukulinganisa ngempumelelo2. Ukukhishwa okunganele kwe-DNA polymerase 3. Umsebenzi wesikhathi eside we-DNA polymerase wehlile | 1. Izinga lokushisa le-anneal liphakeme kunevelu ye-TM ye-primer2. Isikhathi se-pre-denaturation sifushane kakhulu 3. Isikhathi sesigaba ngasinye senqubo yokusabela side kakhulu |
| D. Ukuxuba okunganele kwama-reagents noma amaphutha epayipi | Kusistimu yokusabela, ukugxiliswa kwendawo kwezingxenye zokusabela kwe-PCR kuphezulu kakhulu noma akulingani, okuholela ekukhuliseni okungewona obala kwe-PCR amplification. | |
| E. Ubude be-Amplicon | Ubude be-amplicon bude kakhulu, budlula i-300bp, futhi ukusebenza kahle kokukhulisa kuphansi | Hlola ukuthi ubude be-amplicon buphakathi kuka-80-300bp |
| F. Ithonya lama-reagents e-qPCR | Ukugcwala kwe-DNA polymerase ku-reagent kuphansi noma ukugcwala kwama-ion ku-buffer akuthuthukisiwe, okuholela ekutheni umsebenzi we-enzyme ye-Taq ungafinyeleli ezingeni eliphezulu. | Ukunqunywa kokusebenza kahle kwe-amplification ngejika elijwayelekile |
2.Uhla lwamanani e-Ct
Amanani e-CT asukela ku-15-35.Uma inani le-Ct lingaphansi kuka-15, kucatshangwa ukuthi ukukhuliswa kungaphakathi kwebanga lenkathi eyisisekelo futhi umkhawulo we-fluorescence awukafinyelelwa.Ngokufanelekile, kunobudlelwano bomugqa phakathi kwevelu ye-Ct kanye ne-logarithm yenombolo yokuqala yekhophi yesifanekiso, okungukuthi, ijika elijwayelekile.Ngejika elijwayelekile, lapho ukusebenza kahle kokukhulisa kungu-100%, inani le-Ct elibaliwe lokulinganisa inombolo yekhophi eyodwa yofuzo licishe libe ngu-35. Uma likhulu kuno-35, inombolo yokuqala yekhophi yesifanekiso ngokwengqikithi ingaphansi kuka-1, okungase kubhekwe njengokungasho lutho.
Ngobubanzi obuhlukene bofuzo lwe-Ct, ngenxa yomehluko kunombolo yekhophi yofuzo nokusebenza kahle kokukhulisa inani lesifanekiso sokuqala, kuyadingeka ukwenza ijika elijwayelekile lofuzo futhi kubalwe ububanzi bokutholwa komugqa wofuzo.
3.Izici ezinomthelela yenani le-Ct
Kusukela ebuhlotsheni phakathi kwenani lemijikelezo yokukhulisa kanye nenani lomkhiqizo: inani lomkhiqizo okhulisiwe = inani lesifanekiso sokuqala × (1+En) inombolo yomjikelezo womjikelezo, kungabonakala ukuthi ngaphansi kwezimo ezinhle, inani lesifanekiso sokuqala kanye ne-En kuzoba nomthelela omubi ku-Ct value kuyathinteka.Umehluko kukhwalithi yesifanekiso noma ukusebenza kahle kokukhulisa kuzobangela inani le-Ct ukuthi libe likhulu kakhulu noma libe lincane kakhulu.
Inani le-4.Ct likhulu kakhulu noma lincane kakhulu
Isikhathi sokuthumela: Feb-22-2023
