Ukuhola

I-RNA ende engafaki amakhodi, i-lncRNA iyi-RNA engafaki amakhodi enobude obungaphezu kwama-nucleotide angama-200, ngokuvamile phakathi kuka-200-100000 nt.I-lncRNA ilawula ukubonakaliswa kofuzo kumazinga e-epigenetic, okulotshiweyo kanye nangemuva kokubhala, futhi ibamba iqhaza ekuthuliseni i-chromosome engu-X, ukuphrinta kwe-genome nokuguqulwa kwe-chromatin, ukwenziwa kusebenze kokubhala okulotshiweyo, ukuphazamiseka kokubhala, ukuthuthwa kwenyukliya, ukulawulwa komjikelezo weseli, ukulawulwa kokuhlukaniswa kwamaseli, kanye nezinqubo eziningi zokunxeshezelwa komthamo neminye imiphumela ebalulekile yesinxephezelo (Dosa kanye nezinqubo eziningi zesinxephezelo) ukuvimbela izifo zabantu, futhi njengamanje zingenye yezindawo ezishisayo emkhakheni we-biomedicine.

01  Izinhlobo nezici ze-LncRNA

I-lncRNA ihlukaniswe yaba izinhlobo eziningi ngokusho kwemithombo ehlukene yokwakheka kwayo, njengoba kuboniswe esithombeni esingenhla.Inezici ezilandelayo:

1. Ama-lncRNA avamise ukuba made, anenkulumo eguquguqukayo nezindlela ezihlukene zokuhlanganisa ngesikhathi sokuhlukaniswa
2. Uma kuqhathaniswa nofuzo lwekhodi, i-lncRNA ivamise ukuba nezinga lokusho eliphansi
3. Ama-lncRNA amaningi anencazelo esobala yesikhashana neyendawo enqubweni yokuhlukaniswa kwezicubu nokuthuthuka.
4. Kukhona izinkulumo zesici kumathumba nezinye izifo
5. Izindawo ezingaphansi kwamaselula e-lncRNA zihlukahlukene
6. Ukulandelana kuncane ekongiweni, kodwa umsebenzi unezinga elithile lokonga, njll.

02 Imibuzo Evame Ukubuzwa mayelana ne-LncRNA Reverse Transcription

Ngenxa yokuqukethwe okuphansi kwe-lncRNA kumaseli, ubude obude kanye nesakhiwo sezinga eliphezulu, i-RNA enjalo ivamise ukuba nezakhiwo eziyinkimbinkimbi ezifana namaluphu esiqu noma amaphini ezinwele, futhi i-RT-qPCR ijwayele ukuba nezinkinga zokuloba okungaphumeleli noma ukusebenza kahle okuphansi.

03 Isixazululo se-LncRNA sokuhlehla sokuloba

Emthethweni omaphakathi, inqubo yamagciwane e-RNA asebenzisa eyawo i-reverse transcriptase ukuze ahlanganise i-DNA esebenzisa i-RNA njengesifanekiso ibizwa ngokuthi i-reverse transcription.Inqubo yokusebenzisa i-RNA virus reverse transcriptase ukuze kuhlanganiswe isifanekiso se-cDNA ku-vitro ibizwa ngokuthi ukuloba okuhlanekezelwe.Izinto eziyinhloko ezifakwe ohlelweni lokuloba okuhlanekezelwe: isifanekiso se-RNA, i-reverse transcriptase, ama-primers nezinye izingxenye.

1.RNA isifanekiso

Umthelela wesifanekiso se-RNA ekulotshweni okuhlehlayo ubonakala esakhiweni saso, ubumsulwa nobuqotho.Ukuphakama kobumsulwa nobuqotho be-RNA ekhishiwe, kuphakama ukusebenza kahle kokuhlehliswa kokuloba kanye nemiphumela yobuningi elandelayo enembe kakhudlwana.I-RNA ehlanzwe izithasiselo zokukhipha i-RNA ezivame ukusetshenziswa ngokuvamile iqukethe izinsalela zotshwala nosawoti we-guanidine, anomthelela oqinile wokuvinjelwa kwe-reverse transcriptase.

2.Reverse transcriptase

I-Reverse transcriptase inomthelela omkhulu kuyo yonke isistimu yokuhlehla yokubhala.Izinga lokushisa elifanele le-reverse transcriptase elisetshenziswa kumalabhorethri amaningi ngokuvamile lingu-42°C.Eqinisweni, ukuvulwa kwesakhiwo se-RNA esiphezulu kudinga izinga lokushisa eliphezulu, futhi izidingo ze-reverse transcriptase ziphakeme.

3.Isiqalo

Ama-primers ahlanekezelwe ahlanganisa ama-primer aqondene nofuzo, ama-random primers kanye nama-Oligo dT.Ama-lncRNA amaningi awanayo imisila ye-polyA, futhi kubalulekile ukufanisa kahle iziqalo ezingahleliwe ne-Oligo dT.

4.Ezinye izingxenye

Uma kubhekwa iqiniso lokuthi i-RNA ilula kakhulu ukwehliswa, ukwethulwa kwezingxenye zesistimu yokubhala okuphambene kungancishiswa, okungavimbela ngempumelelo ukufakwa kwe-RNase futhi kuqinisekise ukuqhubeka okushelelayo kokulotshiweyo okuhlehlayo.

Foregeneithola kuthempulethi ye-RNA, hlehlisa ukulotshwa ku-qPCR

Phepha ngokugcwele ukutholwa kwakho kwe-lncRNA

Ingqikithi Yezilwane I-RNA IukuthulaIkhithi (enekholomu yokususwa kwe-gDNA)

Ingqikithi Yeseli RNA IukuthulaIkhithi (enekholomu yokususwa kwe-gDNA)

I-Lnc-RT HeroTM I(With gDNase)(Super Premix for first-strand cDNA synthesis from lncRNA)

Isikhathi Sangempela PCR EasyTM-SYBR Green I

Aizinzuzo ofUkukhishwa kwe-RNA:

Ikhithi isebenzisa ubuchwepheshe bokukhipha i-RNA besizukulwane sesithathu, asikho isidingo sokusebenzisa i-DNase.

Imizuzu eyi-11 ukusuka ku-96, 24, 12, kanye namaseli akhuliswe kahle wepuleti ayi-6, ukukhishwa okuphezulu okusebenzayo kwe-gDNA-free, high-purity, high quality total cell RNA.

Izinzuzo zokulotshwa kokuhlehla kwe-lncRNA:

Isistimu yokubhala okuhlanekezelwe ngokukhethekile eyenzelwe i-LncRNA ukuze isuse ngokushesha ukungcoliswa kwe-genomic DNA.

Isistimu yokubhala ngokuhlehlayo inokuzinza okuphezulu kwe-thermal futhi isenokusebenza okuhle kokuhlehla kokulotshiweyo kokuthi 50°C.

Izifanekiso ezinokuqukethwe okuphezulu kwe-GC kanye nesakhiwo sesibili esiyinkimbinkimbi singahlehliswa ngokusebenza kahle okuphezulu.

Izinzuzoof qI-PCR:

Isiqalo esishisayo i-Foregene Taq Polymerase inekhono eliphezulu lokukhulisa i-amplification, ukuzwela kwe-amplification ephakeme, nokucaciswa kokukhulisa okuphezulu

I-Real PCR Easy Mix ethuthukisiwe yenza i-SYBR Green I ibe nokuzwela okuphezulu kokutholwa, futhi amandla ayo e-fluorescence aphindwe izikhathi ezingu-3-5 kunemikhiqizo efanayo, engahlangabezana nezidingo zezinhlobo ezahlukene zokuhlolwa kobuningi be-fluorescence.