Ƙimar Ct ita ce mafi mahimmancin sakamako na gabatarwa na PCR mai ƙima.Ana amfani da shi don ƙididdige bambance-bambancen maganan kwayoyin halitta ko lambar kwafin halitta.Don haka menene ƙimar Ct na ƙididdigewa mai walƙiya da aka ɗauka m?Yadda za a tabbatar da ingantaccen kewayon ƙimar Ct?
Menene Ct Value?
A yayin aiwatar da haɓakawa na qPCR, adadin madaidaicin ƙararrawar zagayowar (Cycle Threshold) lokacin da siginar walƙiya na haɓakar samfurin ya kai ga saita bakin ƙyalli.C yana nufin Cycle kuma T yana nufin Ƙofa.A taƙaice, ƙimar Ct ita ce adadin zagayowar da ta yi daidai da lokacin da haɓaka samfurin farko ya kai wani adadin samfur a qPCR.Abin da ake kira "wani adadin samfurin" za a ƙara yin bayani daga baya.
Menene ƙimar Ct ke yi?
1. Dangantakar da ke tsakanin haɓakar haɓakawa, adadin samfuri da ƙimar Ct
Mahimmanci, kwayoyin halitta a cikin qPCR ana tara su ta hanyar haɓakawa mai ƙima bayan wasu adadin zagayowar.Dangantakar da ke tsakanin adadin zagayowar haɓakawa da adadin samfuran ita ce: Ƙimar samfur Adadin = adadin samfurin farko × (1+En) lambar hawan keke.Koyaya, halayen qPCR ba koyaushe yana cikin kyakkyawan yanayi ba.Lokacin da adadin ƙaƙƙarfan samfurin ya kai “waɗansu adadin samfuran”, adadin zagayowar a wannan lokacin shine ƙimar Ct, kuma yana cikin lokacin ƙarawa mai ƙima.Dangantakar da ke tsakanin ƙimar Ct da adadin fara samfur: Akwai dangantaka ta layi tsakanin ƙimar Ct na samfuri da logarithm na lambar kwafin farawa na samfuri.Mafi girman ƙaddamarwar samfurin farko, ƙananan ƙimar Ct;ƙananan ƙaddamarwar samfurin farko, mafi girman ƙimar Ct.
2.Amplification curve, fluorescence ƙofa da wasu adadin samfurin PCR
Adadin samfurin ƙarawa na qPCR ana gabatar da shi kai tsaye ta hanyar siginar kyalli, wato, lanƙwan ƙarawa.A farkon matakin PCR, haɓakawa yana ƙarƙashin yanayi mai kyau, adadin hawan keke yana da ƙananan, tarin samfuran ƙananan ƙananan, kuma matakin haske ba zai iya bambanta a fili daga bangon haske ba.Bayan haka, hasken wuta yana ƙaruwa kuma ya shiga lokaci mai ma'ana.Ana iya gano adadin samfurin PCR a wani lokaci lokacin da PCR ya kasance kawai a cikin lokaci mai ma'ana, wanda za'a iya amfani dashi azaman "wani adadin samfurin", kuma za'a iya fitar da abun ciki na farko na samfurin daga wannan.Don haka, ƙarfin siginar kyalli wanda ya yi daidai da takamaiman adadin samfur shine madaidaicin ƙyalli.
A ƙarshen mataki na PCR, lanƙwan ƙarawa baya nuna ƙarawa mai ƙima, kuma yana shiga cikin layin layi da lokacin faranti.
3.Reproducibility na Ct dabi'u
Lokacin da sake zagayowar PCR ya kai lambar sake zagayowar ƙimar Ct, yanzun ya shiga lokacin haɓakawa na gaskiya.A wannan lokacin, ƙananan kuskuren ba a haɓaka ba, don haka sake fasalin darajar Ct yana da kyau, wato, samfurin iri ɗaya yana ƙarawa a lokuta daban-daban ko a cikin tube daban-daban a lokaci guda.Ƙarawa, ƙimar Ct da aka samu akai akai.
1.Amplification yadda ya dace En
Ingantaccen haɓakawa na PCR yana nufin dacewa da abin da polymerase ke canza kwayar halitta don haɓakawa zuwa amplicon.Ingantacciyar haɓakawa lokacin da kwayar halittar DNA ɗaya ta canza zuwa ƙwayoyin DNA guda biyu shine 100%.Ana bayyana ingancin haɓakawa da yawa azaman En.Domin sauƙaƙe nazarin labaran da ke gaba, an gabatar da abubuwan da suka shafi ingancin haɓakawa a takaice.
| Abubuwa masu tasiri | bayani | Yadda za a yi hukunci? |
| A. PCR masu hanawa | 1. Samfurin DNA yana ƙunshe da abubuwa waɗanda ke hana halayen PCR, kamar sunadaran gina jiki ko kayan wanka.2. cDNA bayan juyar da rubutun ya ƙunshi babban taro na samfuri RNA ko RT reagent aka gyara, wanda kuma zai iya hana halayen PCR na gaba. | 1. Ana iya tantance ko akwai gurɓataccen abu ta hanyar auna ma'aunin A260/A280 da A260/A230 ko RNA electrophoresis.2. Ko an narkar da cDNA bisa ga ƙayyadaddun rabo bayan an juyar da rubutun. |
| B. Zane na farko mara kyau | Fim ɗin ba sa gogewa da kyau | Bincika firamare don firamare-dimers ko ginshiƙan gashi, rashin daidaituwa, da kuma wasu lokuta masu faɗin ƙirar ciki. |
| C. Tsarin tsarin amsawar PCR mara kyau | 1. Fim ɗin ba za su iya shafewa yadda ya kamata ba2. Rashin isasshen sakin DNA polymerase 3. Ayyukan DNA polymerase mai girma na dogon lokaci ya ragu | 1. Zazzaɓin zafin jiki ya fi ƙimar TM na firamare2. Lokacin pre-denaturation ya yi guntu sosai 3. Lokacin kowane mataki na hanyar amsawa ya yi tsayi da yawa |
| D. Rashin isasshen hadawa na reagents ko kurakurai na pipetting | A cikin tsarin amsawa, ƙaddamarwar gida na abubuwan abubuwan amsawar PCR ya yi yawa ko rashin daidaituwa, wanda ke haifar da haɓakar haɓakar PCR mara fa'ida. | |
| E. Tsawon Amplicon | Tsawon amplicon ya yi tsayi da yawa, ya zarce 300bp, kuma ingancin ƙarawa ya yi ƙasa kaɗan. | Bincika cewa tsawon amplicon yana tsakanin 80-300bp |
| F. Tasirin qPCR reagents | Matsakaicin DNA polymerase a cikin reagent yana da ƙasa ko kuma ba a inganta yawan ions a cikin buffer ba, wanda ya haifar da aikin enzyme Taq bai kai matsakaicin ba. | Ƙayyadaddun ingancin haɓakawa ta daidaitaccen lanƙwasa |
2.Range na ƙimar Ct
Ƙimar Ct tana daga 15-35.Idan darajar Ct ta kasa da 15, ana la'akari da cewa haɓakawa yana cikin kewayon lokacin tushe kuma ba a kai ga matakin haske ba.Da kyau, akwai alaƙar layi tsakanin ƙimar Ct da logarithm na lambar kwafin farko na samfuri, wato, daidaitaccen lanƙwasa.Ta hanyar ma'auni mai mahimmanci, lokacin da ƙarfin haɓakawa ya kasance 100%, ƙimar Ct da aka ƙididdige don ƙididdige lambar kwafin kwayar halitta yana kusa da 35. Idan ya fi 35 girma, lambar kwafin farko na samfurin ya kasance ƙasa da 1, wanda za'a iya la'akari da rashin ma'ana.
Don nau'ikan jinsin Ct daban-daban, saboda bambancin lambar kwafin kwayoyin halitta da ingancin haɓakawa a cikin adadin samfuri na farko, ya zama dole a yi daidaitaccen lanƙwasa ga kwayar halitta da ƙididdige kewayon gano layin na kwayar halitta.
3.Tasirin abubuwan da ke haifar da ƙimar Ct
Daga alaƙar da ke tsakanin adadin hawan haɓakawa da adadin samfurin: adadin haɓaka samfurin = adadin samfurin farko × (1 + En) lambar sake zagayowar, ana iya ganin cewa a ƙarƙashin yanayi mai kyau, adadin samfurin farko da En zai sami mummunan tasiri akan ƙimar Ct.Bambanci a cikin ingancin samfuri ko ingancin haɓakawa zai sa ƙimar Ct ta zama babba ko ƙarami.
4.Ct darajar yayi girma ko kuma karami
Lokacin aikawa: Fabrairu-22-2023
