Ukuhlolwa kwe-RT-qPCR kufaka phakathi ukukhipha kwe-RNA nokuhlola ikhwalithi, ukuloba okuhlanekezelwe kanye nezinyathelo ezintathu ze-qPCR, isinyathelo ngasinye sinezinyathelo eziningi zokuphepha, sizokwethula ngokuningiliziwe ngezansi.

Ⅰ.Ukuhlolwa kwekhwalithi ye-RNA

Ocwaningweni lwe-RT-qPCR, ngemva kokuqedwa kokukhipha i-RNA, ikhwalithi ye-RNA idinga ukuhlolwa, futhi isilingo sokulandelela singenziwa kuphela ngemva kokuba isifanelekile.Izindlela zokuhlola zihlanganisa i-spectrophotometer, i-Agilent gel electrophoresis, ukuhlaziywa kwe-Agilent 2100, phakathi kwayo okuvame ukusetshenziswa kakhulu i-spectrophotometer kanye ne-agarose gel electrophoresis indlela yokuthola.Kufanele kuqashelwe ukuthi lezi zindlela ezimbili zidinga ukusetshenziswa ndawonye ukuze kuphothulwe ukutholwa nokuhlaziywa kokugxila kwe-RNA, ubumsulwa nobuqotho, ukuze kuqinisekiswe ikhwalithi ye-RNA.

Ikhithi Ehlobene ye-RNA Isolation: 

Ikhithi Yokuzihlukanisa Yeseli Ye-RNA

I-RNA ehlanzwe kakhulu nesezingeni eliphezulu ingatholakala kumaseli ahlukahlukene akhulisiwe ngemizuzu eyi-11.

Ikhithi Yokuhlukanisa Yezilwane Ephelele ye-RNA

Khipha ngokushesha nangempumelelo ukuhlanzeka okuphezulu nekhwalithi ephezulu ingqikithi ye-RNA ezicutshini zezilwane ezahlukahlukene.

I-Spectrophotometer:

I-spectrophotometer isetshenziswa kakhulu ukucacisa ukugxiliswa nokuhlanzeka kwe-RNA, kodwa ayikwazi ukubona ubuqotho be-RNA nezinsalela ze-genomic.Phakathi kwazo, i-A260/280 ne-A260/230 ziyimingcele ebalulekile yokuthola ubumsulwa be-RNA, futhi ubumsulwa be-RNA bungatholwa ngokuya ngokuguquguquka kwamanani abo:

1. 1.9< A260/280< 2.1, okubonisa ukuthi ubumsulwa be-RNA buhle;I-A260/280<1.9, ebonisa ukuthi kungase kube khona insalela yamaprotheni ku-RNA;I-A260/280>2.1, ekhombisa ukucekelwa phansi kwe-RNA okungaba khona, okungaqinisekiswa ngokuqhubekayo yi-agarose gel electrophoresis.

2. 2.0< A260/230< 2.2, okubonisa ukuthi ubumsulwa be-RNA buhle;I-A260/230< 2.0, ebonisa ukuthi kungase kube khona izinsalela zama-organic reagents ku-RNA, njenge-phenols, i-ethanol noma ushukela.

I-Agarose gel electrophoresis:

I-Agarose gel electrophoresis assay ingahlaziya ubuqotho be-RNA, i-genome nezinsalela zamaprotheni, kodwa ayikwazi ukukala ngokunembile ukugcwala kwe-RNA noma ithole izinsalela zama-organic reagents.Thatha izifanekiso ze-eukaryotic RNA ngokwesibonelo:

1. I-RNA yayingaphansi kwe-agarose gel electrophoresis.Uma bekunamabhendi amathathu kuphela okuthi 28sRNA, 18sRNA kanye no-5.8sRNA kumephu yejeli, kubonisa ukuthi i-RNA ekhishiwe iphelele.Uma kukhona into edonsayo, ikhombisa ukucekelwa phansi kwe-RNA ngokwengxenye.

2. Uma kunebhande elilodwa elikhanyayo phakathi kwembobo yeglue kanye nebhendi ye-28sRNA, kungase kube khona izinsalela ze-genomic DNA.

3. Uma amabhande ebonakala emgodini weglue, kubonisa ukuthi kungase kube khona izinsalela zamaprotheni nezinye izinto ze-macromolecular.

Ⅱ. Hlehlisa ukuloba

Ngemva kokuqedwa kokukhipha i-RNA, idinga ukubuyiselwa emuva ku-cDNA ukuze kwenziwe izivivinyo ezilandelayo, ngakho isinyathelo sokuhlehla sibalulekile.Ukuloba okuhlanekezelwe kuzokwethulwa kusukela ekukhethweni kwe-reverse transcriptase kanye ne-primer:

Hlehlisa ukukhetha kwe-transcriptase:

Okulotshiweyo okuhlanekezelwe okujwayelekile kufaka i-AMV RTase ne-MMLV RTase.I-RNase H ye-AMV RTase inomsebenzi oqinile, ubude be-synthesis obufushane, inani eliphansi le-synthesis kanye nokuzinza okuhle kwe-thermal (42 ~ 55℃).Umsebenzi we-RNase H we-MMLV RTase awunamandla, ubude bokuhlanganisa bude, inani le-synthesis liphezulu, futhi ukuzinza kwe-thermal kubi (37 ~ 42℃).

Ngenxa yokuthi i-enzyme ye-RNase H inomsebenzi wokwehlisa isifanekiso se-RNA, i-MMLV enomsebenzi obuthakathaka we-RNase H kufanele ikhethwe ngokukhethekile ngesikhathi sokuloba okuhlanekezelwe, futhi ngemva kobunjiniyela bofuzo bakamuva, ukuzinza okushisayo kwe-MMLV kufinyelele izinga eliphezulu.Ukuthatha ForegeneI-Foreasy Reverse Transcriptase(M-MLV yokubhala okuhlanekezelwe) njengesibonelo, i-reverse transcriptase entsha evezwa kubhaktheriya enziwe nge-E. coli esebenzisa ubuchwepheshe bokuhlanganiswa kabusha kofuzo.Kuyi-recombinant DNA polymerase ehlanganisa umucu we-DNA ohambisanayo kusukela ku-RNA enomucu owodwa, i-DNA, noma i-RNA:ingxube ye-DNA.Ayinawo umsebenzi we-RNase H, ukuzinza okuqinile, ukuhlobana okuqinile kwe-RNA, nokuzwela okuphezulu kokutholwa.

 

I-Foreasy Reverse Transcriptase(M-MLV yokubhala okuhlanekezelwe)

Ukukhetha i-primer:

Ngokuvamile ama-primers e-RT awela ezigabeni ezintathu: i-oligo dT, iziqalo ezingahleliwe, neziqalo eziqondene nofuzo oluthile.Khetha ama-primer afanelekile ongawasebenzisa ngokuya ngezidingo zokuhlola ezahlukahlukene.

1. Uma isifanekiso sinomsuka we-eukaryotic futhi i-cDNA engasekho isetshenziselwa ukukhulisa i-PCR okujwayelekile, i-Oligo (dT) iyanconywa;Uma ukuhlola okulandelayo kusetshenziswa kuphela i-qPCR, i-Oligo (dT) iyanconywa ukuthi ixutshwe neziqalo ezingahleliwe ukuze kuthuthukiswe ukusebenza kahle kokulotshwa okuhlanekezelwe.

2. Uma ithempulethi ivela kuma-prokaryotes, ama-Primer Random noma ama-primer aqondene nofuzo kufanele akhethwe ukuze alotshwe ngokuhlehla.

Ⅲ.qPCR

I-Fluorescence quantification icaciswa ngokuyinhloko ekukhethweni kwezindlela zobuningi, izimiso zokuklama kokuqala, ukukhethwa kwe-ROX, ukucushwa kwesistimu yokusabela kanye nokusetha izimo zokusabela, njll.

Ukukhetha izindlela zobuningi:

Izindlela ze-quantitative zihlukaniswe zibe izindlela zokuqhathanisa nezindlela zobuningi obuphelele.Ukulinganisa okuhlobene kungasetshenziswa ukuthola umthelela wezindlela ezithile zokwelapha ekukhulumeni kwezakhi zofuzo, ukubona umehluko wokuvezwa kwezakhi zofuzo ngezikhathi ezihlukene futhi kuqhathaniswe umehluko wokuvezwa kofuzo ezicutshini ezihlukene.I-Absolute quantification ingathola inani le-nucleic acid egciwaneni nokunye.Lapho senza izivivinyo, kufanele sikhethe izindlela zobuningi ezifanele ngokuya ngokuhlolwa kwethu.

Izimiso zomklamo wokuqala:

Idizayini ye-primer ye-qPCR ihlobene ngqo nokusebenza kahle kwe-amplification kanye nemininingwane yomkhiqizo.Ngakho-ke, ukuklama kahle iziqalo ezinhle kuyisinyathelo sokuqala se-qPCR eyimpumelelo.Ekwakhiweni kwe-primer, izimiso ezilandelayo kufanele zinakwe lapho kuhlangatshezwana nesimiso somklamo ovamile we-primer:

1. Ubude bocezu oluqondiwe kulawulwa phakathi kuka-100 no-300 bp;

2. Idizayini ye-cross-exon yokugwema ithonya le-genomic DNA;

3. Ama-primer aklanyelwe kudingeka ahlolelwe ukusebenza kahle kokukhulisa, futhi kuphela uma ukuphumelela kokukhulisa kufika ezingeni (90-110%) lapho kungasetshenziselwa ukuhlola ubuningi;

4. I-Primer concentration ivamise ukulungiselelwa phakathi kuka-0.1uM no-1.0uM.

Ukukhethwa kweI-ROX:

Enqubweni yokusabela kwenani, i-ROX ingalungisa umehluko wendlela yokubona, iphutha lepayipi noma umehluko wevolumu obangelwa ukuhwamuka nokufiphala ngendlela efanayo, ithuthukise ukuphindaphinda kwemiphumela.Kodwa-ke, kufanele kuqashelwe ukuthi ukukhethwa kwe-ROX kuhlobene nethuluzi.Uma insimbi ye-qPCR inomsebenzi wokulungisa ngokuzenzakalelayo umehluko phakathi kwezimbobo, ayidingi ukwengeza i-ROX;ngaphandle kwalokho, idinga ukwengeza ukulungiswa kwe-ROX.Ozakwethu abancane ekuthengeni ama-reagents kumele bahambisane nethuluzi elisetshenziswa ukukhetha i-ROX efanele, gwema amaphutha akamuva.

Ukulungiswa kwesistimu yokusabela:

Amavolumu okusabela angama-20ul kanye nama-50ul ayakhethwa.Izinto ezilandelayo kufanele zinakwe lapho kwakhiwa uhlelo:

1. Isistimu yokusabela idinga ukulungiswa ngokungenisa umoya ebhentshini lokusebenza elihlanzekile kakhulu, i-ddH entsha₂O isetshenziselwa ukuhlola ngakunye;

2. Ukuhlolwa ngakunye kudinga ukulungiselela i-NTC ukuze iqinisekise ukuthi kukhona yini ukungcola ohlelweni, futhi zonke iziqalo kufanele zenze i-NTC lapho kulungiselelwa uhlelo;

3. Ukuthola ukuthi ingabe ikhona yini insalela ye-gDNA kusifanekiso se-RNA, i-NRT ingalungiselelwa isampula ngayinye ukuze itholwe;

4. Lapho ulungiselela uhlelo, kunconywa ukwenza okungenani ukuphindaphinda kwezobuchwepheshe oku-3 kwesampula eyodwa;

5. Uma ithempulethi kuyi-cDNA, kuyatuswa ukuthi kuhlanjululwe izikhathi ezi-5-10 ukuze kwehliswe umthelela wokuvimbela wesistimu yokubhala okuhlanekezelwe ekuhlolweni kwe-qPCR.Kungcono ukuhlola ubuningi besifanekiso nge-gradient, ukuze inani le-CT liwele phakathi kuka-20-30;

6. Thola inombolo edingekayo yokusabela, ukwandisa ngo-5-10% ngesisekelo senani lokusabela, futhi ubale inombolo yokumisa ivolumu;

7, uhlelo lulungiswa kusetshenziswa umgomo we-premix, ukuxuba ngemuva kwe-centrifugation futhi kuqinisekiswe ukuthi awekho amabhamuza;

8, Ngokusemandleni ukukhetha izinto ezisetshenziswayo ezisekelayo.

Ikhithi ye-RT-qPCR ehlobene