1. Ulwazi oluyisisekelo (uma ufuna ukubona ingxenye yokuhlola, sicela udlulisele ngqo engxenyeni yesibili)
Njengokusabela kokuphuma kokunye kwe-PCR evamile, I-PCR yesikhathi sangempela iqapha ngokuyinhloko ukuguqulwa kwenani lomkhiqizo wokukhulisa izwi kumjikelezo ngamunye wokusabela kokukhulisa i-PCR ngesikhathi sangempela ngoshintsho lwesignali ye-fluorescence, futhi ihlaziya ngokobuningi isifanekiso sokuqala ngobudlelwano phakathi kwevelu ye-ct kanye nejika elijwayelekile .
Imininingwane ethile ye-RT-PCR yileisisekelo, ukukhanya kwe-fluorescencefuthiInani le-CT.
| isisekelo: | Inani le-fluorescence lomjikelezo wesi-3-15 liyisisekelo (isisekelo), esibangelwa iphutha lezikhathi ezithile lokulinganisa. |
| I-Threshold (i-threshold): | Isho umkhawulo wokutholwa kwe-fluorescence obekwe endaweni efanele endaweni yokukhula okucacile yejika lokukhulisa, ngokuvamile izikhathi ezingu-10 ukuchezuka okujwayelekile kwesisekelo. |
| Inani le-CT: | Yinombolo yemijikelezo ye-PCR lapho inani le-fluorescence kushubhu ngalinye lokusabela lifinyelela embundwini. Inani le-Ct liphambene ngokuphambene nenani lesifanekiso sokuqala. |
Izindlela zokulebula ezijwayelekile ze-RT-PCR:
| indlela | inzuzo | ukushiyeka | ububanzi besicelo |
| I-SYBR GreenⅠ | Ukusebenziseka okubanzi, okubucayi, okushibhile futhi kulula | Izidingo zokuqala ziphezulu, zithambekele kumabhendi angacacisiwe | Ilungele ukuhlaziya inani lezakhi zofuzo eziqondiwe ezihlukahlukene, ucwaningo mayelana nokuvezwa kwezakhi zofuzo, kanye nocwaningo lwezilwane nezitshalo ezingashintshiwe. |
| TaqMan | Ukucaciswa okuhle nokuphindaphinda okuphezulu | Intengo iphezulu futhi ilungele imigomo ethile kuphela. | Ukutholwa kwe-pathogen, ucwaningo lofuzo olungazweli emuthini, ukuhlola ukusebenza kwezidakamizwa, ukuhlonzwa kwezifo zofuzo. |
| isibani samangqamuzana | Ukucaciswa okuphezulu, i-fluorescence, ingemuva eliphansi | Intengo iphakeme, ifaneleka kuphela injongo ethile, umklamo unzima, futhi intengo iphakeme. | Ukuhlaziywa kofuzo okuqondile, ukuhlaziywa kwe-SNP |
2. Izinyathelo zokuhlola
2.1 Mayelana neqoqo lokuhlola- kufanele kube nemithombo eminingi eqenjini, futhi kufanele kube nokuphindaphinda kwezinto eziphilayo.
| ① | Ukulawula okungenalutho | Isetshenziselwa ukuthola isimo sokukhula kweseli ekuhlolweni |
| ② | Ukulawula okungalungile kwe-siRNA (ukulandelana kwe-siRNA okungaqondile) | Bonisa ukucaciswa kwesenzo se-RNAi.I-siRNA ingase ibangele impendulo yokucindezeleka engaqondile ekugxilweni kwe-200nM. |
| ③ | Ukulawulwa kwe-Reagent yokudlulisa | Ungafaki ubuthi be-reagent transfection kumaseli noma umthelela ekuvezweni kofuzo oluqondiwe |
| ④ | i-siRNA ngokumelene nofuzo oluqondiwe | Shayela phansi isisho sofuzo oluqondiwe |
| ⑤ (uma uthanda) | i-siRNA enhle | Isetshenziselwa ukuxazulula izinkinga zesistimu yokuhlola kanye nezinkinga zokusebenza |
| ⑥ (uma uthanda) | I-Fluorescent control siRNA | Ukusebenza kahle kokudluliswa kweseli kungabonwa ngesibonakhulu |
2.2 Izimiso zomklamo wokuqala
| Usayizi wesiqephu okhulisiwe | Okungcono kakhulu ku-100-150bp |
| Ubude be-Primer | 18-25bp |
| Okuqukethwe kwe-GC | 30% -70%, okungcono 45% -55% |
| Tm inani | 58-60℃ |
| Ukulandelana | Gwema i-T/C eqhubekayo;I-A/G iyaqhubeka |
| 3 ukuphela ngokulandelana | Gwema i-GC ecebile noma i-AT ecebile;isisekelo setheminali kungcono kakhulu u-G noma u-C;kungcono ukugwema u-T |
| Ukuhambisana | Gwema ukulandelana okulandelanayo kwezisekelo ezingaphezu kuka-3 ngaphakathi kwe-primer noma phakathi kwama-primer amabili |
| Ukucaciswa | Sebenzisa usesho lokuqhuma ukuze uqinisekise ukucaciswa kokuqala |
①SiRNA incike kuhlobo oluthile, futhi ukulandelana kwezinhlobo ezahlukene kuzohluka.
②I-SiRNA ipakishwe kumpushana oqandisiwe, ongagcinwa ngokuqinile amaviki angu-2-4 ekamelweni lokushisa.
2.3 Amathuluzi noma ama-reagents adinga ukulungiswa kusenesikhathi
| I-Primer (inkomba yangaphakathi) | Kuhlanganisa phambili nokuhlehla okubili |
| Ama-Primers (ufuzo oluqondiwe) | Kuhlanganisa phambili nokuhlehla okubili |
| I-Target Si RNA (imichilo emi-3) | Ngokuvamile, inkampani izohlanganisa imichilo emi-3, bese ikhetha eyodwa kwezintathu nge-RT-PCR |
| Ikhithi yokudlulisa | Lipo2000 njll. |
| I-RNA Rapid Isizinda Kit | Okokukhishwa kwe-RNA ngemva kokudluliselwa |
| Ikhithi yokubhala ehlehla esheshayo | ye-cDNA synthesis |
| PCR Amplification Kit | 2×Super SYBR Green I-qPCR Master Mix |
2.4 Mayelana nezindaba ezidinga ukunakwa ezinyathelweni ezithile zokuhlola:
① Inqubo yokudlulisa ye-siRNA
1. Ngokucwenga, ungakhetha ipuleti elinomthombo ongu-24, ipuleti lemithombo engu-12 noma ipuleti lomthombo elingu-6 (isilinganiso sokugxiliswa kwe-RNA esihlongozwayo emthonjeni ngamunye wepuleti elinomthombo wama-24 cishe singu-100-300 ng/uL), futhi ukuminyana kokudluliswa kwamaseli okuphelele kufika ku-60 % -80% noma kunjalo.
2. Izinyathelo zokudlulisela kanye nezidingo ezithile zihambisana ngokuqinile nemiyalelo.
3. Ngemva kokudlulisela, amasampula angaqoqwa phakathi kwamahora angu-24-72 ukuze kutholwe i-mRNA (RT-PCR) noma ukutholwa kwamaprotheni phakathi kwamahora angu-48-96 (WB)
② Inqubo yokukhipha i-RNA
1. Vimbela ukungcola ngama-enzyme angaphandle.Kubandakanya ikakhulukazi ukugqoka imaski namagilavu ngokuqinile;usebenzisa amathiphu e-pipette afakwe inzalo namashubhu e-EP;amanzi asetshenziswe ekuhloleni kumele Angabi-RNAse.
2. Kunconywa ukwenza kabili njengoba kuphakanyiswe kukhithi yokukhipha ngokushesha, okuzokwenza ngcono ngempela ukuhlanzeka kanye nesivuno.
3. Uketshezi oluwudoti akumele luthinte ikholomu ye-RNA.
③ ukulinganisa kwe-RNA
Ngemuva kokuthi i-RNA ikhishiwe, ingalinganiswa ngokuqondile ne-Nanodrop, futhi ukufundwa okuncane kungase kube phansi njengo-10ng/ul.
④Hlehlisa inqubo yokubhala
1. Ngenxa yokuzwela okuphezulu kwe-RT-qPCR, kufanele kwenziwe okungenani imithombo emi-3 ehambisanayo kusampula ngayinye ukuvimbela i-Ct elandelayo ukuthi ihluke kakhulu noma i-SD ukuthi ibe nkulu kakhulu ukuze kuhlaziywe izibalo.
2. Ungaqandi futhi uncibilikise I-Master mix ngokuphindaphindiwe.
3. Ishubhu/imbobo ngayinye kufanele kushintshwe ithiphu entsha!Ungasebenzisi ngokuqhubekayo ithiphu elifanayo le-pipette ukwengeza amasampula!
4. Ifilimu enamathiselwe kupuleti lomthombo we-96 ngemuva kokwengeza isampula idinga ukushelela ngepuleti.Kungcono ukuyifaka i-centrifuge ngaphambi kokuyibeka emshinini, ukuze uketshezi olusodongeni lweshubhu lwehle lukhiphe amabhamuza omoya.
⑤Ukuhlaziywa kwejika okujwayelekile
| Asikho isikhathi sokukhula kwe-logarithmic | Ukugxiliswa okukhulu kwesifanekiso okungenzeka |
| Alikho inani le-CT | Izinyathelo ezingalungile zokuthola amasignali e-fluorescent; ukuwohloka kwama-primers noma ama-probes - ubuqotho bayo bungabonwa nge-PAGE electrophoresis; inani elinganele lesifanekiso; ukucekelwa phansi kwezifanekiso - ukugwema ukwethulwa kokungcola kanye nokuqandisa okuphindaphindiwe nokuncibilika ekulungiseni isampula; |
| Ct>38 | Ukusebenza kahle kwe-amplification;Umkhiqizo we-PCR mude kakhulu;izingxenye ezihlukahlukene zokusabela zonakalisiwe |
| Ijika lokukhulisa umugqa | Ama-probe angehliswa kancane imijikelezo yokuncibilika ephindaphindayo noma ukuchayeka ekukhanyeni isikhathi eside. |
| Umehluko ezimbobeni eziyimpinda mkhulu kakhulu | Isixazululo sokusabela asincibilikisiwe ngokuphelele noma isisombululo sokusabela asixubene;ukugeza okushisayo kwensimbi ye-PCR kungcoliswe izinto ze-fluorescent |
2.5 Mayelana nokuhlaziywa kwedatha
Ukuhlaziywa kwedatha ye-qPCR kungahlukaniswa ngobuningi obuhlobene kanye nobuningi obuphelele.Isibonelo, amaseli eqenjini lokwelapha uma kuqhathaniswa namaseli eqenjini elilawulayo,
Ishintsha kangaki i-mRNA yofuzo luka-X, lokhu ukulinganisa okulinganiselwe;enanini elithile lamaseli, i-mRNA yofuzo lwe-X
Mangaki amakhophi akhona, lokhu ukulinganisa okuphelele.Ngokuvamile esikusebenzisa kakhulu elabhorethri indlela yobuningi obuhlobene.Ngokuvamile,indlela ye-2-ΔΔctisetshenziswa kakhulu ekuhlolweni , ngakho le ndlela kuphela ezokwethulwa ngokuningiliziwe lapha.
I-2-ΔΔct indlela: Umphumela otholiwe umehluko ekuvezweni kofuzo oluqondiwe eqenjini lokuhlola elihlobene nofuzo oluqondiwe eqenjini lokulawula.Kudingeka ukuthi ukusebenza kahle kokukhulisa kokubili kofuzo oluqondiwe kanye nofuzo lwangaphakathi lwereferensi kusondele ku-100%, futhi ukuchezuka okuhlobene akufanele kudlule u-5%.
Indlela yokubala imi kanje:
Iqembu lokulawula le-Δct = inani le-ct ye-target gene eqenjini lokulawula - ct value yofuzo lwangaphakathi lwereferensi eqenjini lokulawula
Iqembu lokuhlola le-Δct = inani le-ct yofuzo oluqondiwe eqenjini lokuhlola - ct value yofuzo lwangaphakathi lwereferensi eqenjini lokuhlola
ΔΔct=Δct test group-Δct control group
Okokugcina, bala ukuphindwaphindwa komehluko ezingeni lesisho:
Shintsha iFold=2-ΔΔct (okuhambisana nomsebenzi we-excel AMANDLA)
Imikhiqizo ehlobene:
Ikhithi ye-Cell Direct RT-qPCR
Isikhathi sokuthumela: May-20-2023
