Mayelana nokukhishwa kwe-RNA

Eminyakeni engamashumi amathathu edlule, ekukhishweni kwe-RNA, ebesingakucabanga kwakuwukuthola kuphela i-RNA ephelele, nokho, kwakungekho mfuneko yesivinini sokukhipha (I-Techology yesizukulwane sokuqala indlela ye-Trizol eyazalwa).Ngokuvuselelwa okuphindaphindiwe kobuchwepheshe bokukhipha i-RNA, esingakwenza akukhawulelwe ekutholeni imiphumela.Lapho bebhekene nokukhishwa kwama-reagents asekelwe esizukulwaneni sokuqala se-Trizol, abahloli bavame ukukhathazwa izinyathelo zabo zokusebenza ezinzima;nakuba ngemva kokulungiselelwa okuningi nokuqanjwa kabusha, kusathatha isikhathi esiningi.Ngasikhathi sinye, ama-organic reagents afana ne-phenol ne-chloroform adinga ukusetshenziswa enqubweni yokukhipha, ephinde isongele ukuphepha kwabasebenzi abafanelekile ngokwezinga elithile.

Esebenza kahle, elula futhi ephephile

Ukuthola umkhiqizo okhishwayo we-RNA osebenza kahle, olula futhi ophephile sekuwumgomo oyinhloko wabahloli.Ngakho-ke, indlela ye-Silica Spin Column adsorption yokukhipha i-RNA yaba khona.Imikhiqizo yokukhipha i-RNA yesizukulwane sesithathu se-Foregene idonsa ezinzuzweni zemikhiqizo yokukhipha ikholomu yesizukulwane sangaphambilini, kusetshenziswa(i-DNA-Cleaning + RNA kuphela) ubuchwepheshe obubili be-Spin Column ukuze kukhishwe i-gDNA kanye nemfucumfucu yezicubu ngesikhathi esisodwa, ngaphandle kokwelashwa kwe-DNase kwe-gDNA esele, futhi ingasasebenzisi i-phenol, njll. Izinto ezinobuthi neziyingozi, ubungozi obuncane, isikhathi esifushane se-RNA ukuze kuzuzwe isivuno esihle ne-RNA.

Ishadi lokuqhathanisa lenqubo yokusebenza

Ishadi lokuqhathanisa imiphumela yokuhlola

Ikhithi yokuhlukanisa i-RNA ye-Foregene inesivuno esiphezulu sokukhishwa kwe-RNA, ukuhlanzeka okuhle, kanye nomphumela wokukhipha we-miRNA ungcono kunezinye izinto zomkhiqizo.

I-Electrophoresis ye-50 mg ye-RNA yesibindi segundane esikhishwe ngezindlela ezahlukene
Isizukulwane sokuqala (1: Trizol)
Isizukulwane sesibili (2: Ikhithi yokukhipha i-RNA enkampanini A)
Isizukulwane sesithathu (3: Ikhithi yokukhipha i-RNA kanye nenkampani A, 4: Foregene:RNA Isolation kits)

Igrafu ye-qPCR (igrafu yejika yokukhulisa)

I-Genomic DNA kufanele isuswe ngokuphumelelayo phakathi nokukhipha i-RNA, ngaphandle kwalokho izoba nemiphumela engalawuleki ekuhloleni okuphansi komfula (inani le-CT lokuqala, imibono engamanga).Ikhithi yesizukulwane esilandelayo ye-Foregene ingasusa ngokuphelele i-DNA ye-genomic ngaphandle kokwengezwa kwe-DNase, konga isikhathi esiningi, kuyilapho igwema ukucekelwa phansi okubangelwa ukungcola kwe-RNase yangaphandle.
Isizukulwane sokuqala (okuluhlaza kubonisa i-TRlzol-, okubomvu kubonisa i-TRlzol+)

Isizukulwane sesibili (Okuluhlaza okwesibhakabhaka kukhombisa Ikhithi yokukhipha i-RNA yeNkampani -, okubomvu kubonisa Inkampani Ikhithi yokukhipha i-RNA +)

Isizukulwane sesithathu (oluhlaza okwesibhakabhaka kukhombisa inkampani A RNA extraction kit plus, green ikhombisa Foregene RNA Isolation kits)

(Qaphela: “_” kugrafu ye-qPCR ibonisa ukuthi i-DNase ayizange yengezwe, futhi ukungcoliswa kwe-DNA akukasuswa; “+” kubonisa ukuthi i-DNase yengeziwe, futhi ukungcoliswa kwe-DNA kususiwe)

Qedela ukukhishwa kwe-RNA ngaphakathi kwe-11min

Asikho isidingo seminyaka yokunqwabelana kokuhlangenwe nakho kokukhipha i-nucleic acid, imikhiqizo ye-Foregene ephezulu yokusebenza kahle okuphelele kochungechunge lwekhithi yokuhlukanisa ye-RNA ingakuvumela ukuthi uthole i-RNA enesivuno esikhulu nehlanzeke kakhulu kungakapheli imizuzu eyi-11 ukuze uhlangabezane nezidingo ezahlukahlukene zokuhlola ezansi nomfula.

Kuze kube manje, imikhiqizo yochungechunge lwe-Foregene isiyonke ye-RNA isolaton kits ibonwe inani elikhulu lamakhasimende futhi ibe negalelo ezincwadini eziningi ezinamagoli aphezulu.Ukusetshenziswa kwangempela kwekhasimende kweshadi le-electrophoresis lempendulo kanye nejika lokukhulisa i-PCR ngemva kokuloba okuhlanekezelwe kubonisa ukuthi i-RNA ekhishwe ngokusetshenziswa kwemikhiqizo ye-Foregene ayigcini nje ngokugxila kakhulu, kodwa futhi inobuqotho obuhle.Ubuchwepheshe bomkhiqizo kuphela obuhlolwe yimakethe obungathandwa yiwo wonke umuntu.

Ikhithi Yokuzihlukanisa Yeseli Ye-RNA

Inani eliphakeme kakhulu lama-RNA

Ngokushesha: Qeda Ukuhlukaniswa kwe-RNA ngemizuzu eyi-11

Ukuphepha: Awekho amakhemikhali e-Organic angeziwe

Ezinye izingcaphuno zamaphuzu aphezulu:

1. Yuan Fang, Zezhong Liu, Yang Qiu, et al.Ukuvinjelwa kwe-viral suppressor yamaprotheni e-RNAi ngama-peptide omklami kuvikela ekuthelelekeni kwe-enteroviral ku-vivo, Immunity, 54(10), 2021: 2231-2244.e6.(IF:31.745,Ikhithi yokuhlukanisa i-Viral RNA,Cat No.RE-02011)

https://www.sciencedirect.com/science/article/pii/S1074761321003617

2. URen, Y., Wang, A., Wu, D. et al.Ukuvinjelwa okukabili kokuzivikela komzimba okungokwemvelo kanye ne-apoptosis ngephrotheni yomuntu ye-cytomegalovirus UL37x1 kwenza ukuphindaphinda kwegciwane okuphumelelayo.I-Nat Microbiol 7, 1041–1053 (2022).(IF:30.964,Ikhithi Yokuzihlukanisa Yeseli Ye-RNA,Cat No.RE-03111)

https://doi.org/10.1038/s41564-022-01136-6