Ixabiso le-CT yeyona ndlela ibalulekileyo yokubonisa isiphumo se-fluorescent quantitative PCR.Isetyenziselwa ukubala umahluko kwintetho yemfuza okanye inombolo yekopi yemfuza.Ke lithini ixabiso le-Ct le-fluorescence quantification ethathwa njengefanelekileyo?Indlela yokuqinisekisa uluhlu olusebenzayo lwexabiso le-Ct?
Yintoni ixabiso le-CT?
Ngexesha lenkqubo yokukhulisa i-qPCR, inani elihambelanayo lemijikelo yokukhulisa (Cycle Threshold) xa isignali ye-fluorescence yemveliso eyandisiweyo ifikelela kumda omiselweyo we-fluorescence.UC umele iCycle kwaye uT umele iThreshold.Ukubeka nje, ixabiso le-Ct linani lemijikelezo ehambelanayo xa i-template yokuqala yokukhulisa i-template ifikelela kwisixa esithile semveliso kwi-qPCR.Okubizwa ngokuba "yinani elithile lemveliso" liya kuchazwa ngakumbi kamva.
Lenza ntoni ixabiso le-Ct?
1.Ubudlelwane phakathi kwe-exponential amplification, isixa setemplate kunye nexabiso le-Ct
Ngokufanelekileyo, iijini kwi-qPCR ziqokelelwa ngokwandisa umthamo emva kwenani elithile lemijikelo.Ubudlelwane phakathi kwenani lemijikelo yokukhulisa kunye nenani leemveliso yile: Isixa semveliso eyandisiweyo = isixa sokuqala setemplate × (1+En) inombolo yemijikelo.Nangona kunjalo, impendulo ye-qPCR ayisoloko ikwimeko efanelekileyo.Xa inani lemveliso ekhulisiwe lifikelela "kwixabiso elithile lemveliso", inani lemijikelezo ngeli xesha lixabiso le-Ct, kwaye likwixesha lokukhulisa i-exponential.Ubudlelwane phakathi kwexabiso le-Ct kunye nesixa se-template yokuqalisa: Kukho ubudlelwane bomgca phakathi kwexabiso le-Ct ye-template kunye ne-logarithm yenombolo yokuqala yekopi ye-template.Okuphezulu koxinzelelo lwetemplate yokuqala, incinci ixabiso le-Ct;esezantsi yoxinaniso lokuqala template, elikhulu Ct ixabiso.
I-2.Ijiko lokukhulisa, i-fluorescence threshold kunye nexabiso elithile lemveliso yePCR
Ubungakanani bemveliso yokukhulisa i-qPCR iboniswe ngokuthe ngqo ngohlobo lophawu lwefluorescent, oko kukuthi, ijiko lokukhulisa.Kwinqanaba lokuqala le-PCR, ukukhulisa kuphantsi kweemeko ezifanelekileyo, inani lemijikelezo lincinci, ukuqokelela kwemveliso kuncinci, kwaye inqanaba le-fluorescence alikwazi ukuhlukaniswa ngokucacileyo kwimvelaphi ye-fluorescence.Emva koko, i-fluorescence iyanda kwaye ingena kwisigaba se-exponential.Ubungakanani bemveliso ye-PCR inokubonwa kwinqanaba elithile xa i-PCR isabela nje kwisigaba esicacileyo, esinokuthi sisetyenziswe "njengexabiso elithile lemveliso", kwaye umxholo wokuqala wethemplate unokunqunyulwa kule nto.Ngoko ke, ubukhulu besignali ye-fluorescence ehambelana nomlinganiselo othile wemveliso ngumda we-fluorescence.
Kwinqanaba lamva le-PCR, igophe lokukhulisa alisabonisi ulwandiso lwe-exponential, kwaye lingena kwisigaba somgca kunye nesigaba seplateau.
3.Ukuveliswa kwakhona kwamaxabiso e-Ct
Xa umjikelezo we-PCR ufikelela kwinani lomjikelo wexabiso le-Ct, lisanda kungena kwixesha lokwenyani lokukhulisa i-exponential.Ngeli xesha, iphutha elincinci alizange likhuliswe, ngoko ukuphindaphinda kwexabiso le-Ct lihle kakhulu, oko kukuthi, i-template efanayo ikhuliswe ngamaxesha ahlukeneyo okanye kwiibhubhu ezahlukeneyo ngexesha elifanayo.Ukukhulisa, ixabiso le-Ct elifunyenweyo lihlala lihleli.
1.Ukwandisa ukusebenza kakuhle En
Ubuchule bokukhulisa i-PCR bubhekisa ekusebenzeni kakuhle apho i-polymerase iguqula ijini ukuze yandiswe ibe yiamplicon.Ubuchule bokukhulisa xa imolekyuli enye yeDNA iguqulwa ibe ziimolekyuli ezimbini zeDNA yi100%.Ukusebenza kokwandisa ngokuqhelekileyo kubonakaliswa njenge-En.Ukuze kube lula ukuhlalutya amanqaku alandelayo, izinto ezichaphazela ukusebenza kakuhle kokukhulisa zingeniswa ngokufutshane.
| Imiba enempembelelo | ingcaciso | Indlela yokugweba? |
| A. PCR inhibitors | 1. I-template ye-DNA iqulethe izinto ezivimbela ukuphendula kwe-PCR, njengeeprotheni okanye ii-detergents.2. I-cDNA emva kwe-reverse transcription iqulethe i-high concentration ye-template RNA okanye i-RT reagent components, enokuthi ithintele ukusabela kwe-PCR okulandelayo. | 1. Ingaba kukho ukungcola kunokugwetywa ngokulinganisa umlinganiselo we-A260 / A280 kunye ne-A260 / A230 okanye i-RNA electrophoresis.2. Ingaba i-cDNA ihlanjululwe ngokomlinganiselo othile emva kokukhutshelwa umva. |
| B. Uyilo olungafanelekanga lwe-primer | Iiprimers azihlali ngokufanelekileyo | Jonga iiprimers zeprimer-dimers okanye hairpins, mismatch, kwaye ngamanye amaxesha ithatha uyilo lwe-intronic. |
| C. Uyilo lwenkqubo yokusabela yePCR engafanelekanga | 1. Ii-primers azikwazi ukucima ngokufanelekileyo2. Ukukhutshwa okunganeleyo kwe-DNA polymerase 3. Ubushushu bexesha elide lomsebenzi we-DNA polymerase wehlile | 1. Ukushisa kwe-annealing kuphezulu kunexabiso le-TM ye-primer2. Ixesha le-pre-denaturation lifutshane kakhulu 3. Ixesha lesigaba ngasinye senkqubo yokusabela lide kakhulu |
| D. Ukuxuba okungonelanga kwee-reagents okanye iimpazamo zepayipi | Kwinkqubo yokusabela, ugxininiso lwasekuhlaleni lwamacandelo e-PCR reaction luphezulu kakhulu okanye alulingani, okukhokelela kulwandiso olungenambonakaliso lokwandiswa kwe-PCR. | |
| E. Ubude beAmplicon | Ubude be-amplicon bude kakhulu, budlula i-300bp, kwaye ukusebenza kakuhle kwe-amplification kuphantsi. | Khangela ukuba ubude be-amplicon buphakathi kwe-80-300bp |
| F. Impembelelo yeerejenti ze-qPCR | Ukuxinwa kwe-DNA polymerase kwi-reagent iphantsi okanye ukuxinwa kwee-ion kwi-buffer akwenziwanga kakuhle, okukhokelela ekubeni umsebenzi we-enzyme ye-Taq ungafikeleli phezulu. | Ukumiselwa kobuchule bokukhulisa ngegophe eliqhelekileyo |
2.Uluhlu lwamaxabiso e-Ct
Amaxabiso e-CT avela kwi-15-35.Ukuba ixabiso le-Ct lingaphantsi kwe-15, kucatshangelwa ukuba i-amplification ingaphakathi kwinqanaba lexesha elisisiseko kwaye i-fluorescence threshold ayifikanga.Ngokufanelekileyo, kukho ubudlelwane bomgca phakathi kwexabiso le-Ct kunye nelogarithm yenombolo yokuqala yekopi ye template, oko kukuthi, ijika eliqhelekileyo.Ngokusebenzisa i-curve esemgangathweni, xa i-amplification ye-amplification i-100%, ixabiso le-Ct elibaliweyo lokulinganisa inani lekopi enye ye-gene lijikeleze i-35. Ukuba ikhulu kune-35, inombolo yokuqala yekopi ye-template ithiyori ingaphantsi kwe-1, enokuthi ithathelwe njengento engenamsebenzi.
Kwiintlobo ezahlukeneyo ze-Ct ye-gene, ngenxa yokwahlukana kwinombolo yekopi ye-gene kunye nokusebenza kakuhle kwe-amplification kwisixa sokuqala se-template, kuyimfuneko ukwenza i-curve esemgangathweni ye-gene kwaye ubale uluhlu lokufumanisa umgca we-gene.
3.Iimpembelelo zexabiso le-Ct
Ukususela kubudlelwane phakathi kwenani lemijikelezo yokukhulisa kunye nenani lemveliso: inani lemveliso eyandisiweyo = inani le template yokuqala × (1 + En) inombolo yomjikelezo, kunokubonwa ukuba phantsi kweemeko ezifanelekileyo, inani le-template yokuqala kunye ne-En iya kuba nefuthe elibi kwixabiso le-Ct lichaphazelekayo.Umahluko kumgangatho wetemplate okanye ukusebenza kakuhle kokukhulisa kuya kubangela ukuba ixabiso leCt libe likhulu kakhulu okanye lincinci.
Ixabiso le-4.Ct likhulu kakhulu okanye lincinci kakhulu
Ixesha lokuposa: Feb-22-2023
