Kuyobora

RNA ndende idafite code, lncRNA ni RNA idafite code ifite uburebure burenga 200 nucleotide, muri rusange hagati ya 200-100000 nt.lncRNA igenga imiterere ya gene kurwego rwa epigenetike, iyandikwa ndetse na nyuma yinyandiko-mvugo, kandi igira uruhare mu gucecekesha X chromosome, gucapa genome no guhindura chromatine, gukora transcript, kwandikirana, gutwara ibinyabiziga bya kirimbuzi, kugenzura ingirabuzimafatizo, no kugabanya ingaruka z’indishyi (ingaruka z’indishyi ziterwa na virusi) hamwe n’ibindi bikorwa byinshi by’ingenzi bigenga ingaruka ziterwa no kubaho, iterambere no gukumira indwara.

01  Ubwoko n'ibiranga LncRNA

lncRNA igabanijwemo ubwoko bwinshi ukurikije amasoko atandukanye yo gushingwa, nkuko bigaragara ku gishushanyo kiri hejuru.Ifite ibi bikurikira:

1. lncRNAs mubisanzwe ni birebire, hamwe nimvugo ifite imbaraga nuburyo butandukanye bwo gutandukanya mugihe cyo gutandukana
2. Ugereranije na coding genes, lncRNA mubisanzwe ifite urwego rwo hasi rwo kwerekana
3. LncRNAs nyinshi zifite imiterere yigihe gito nigitekerezo cyihariye muburyo bwo gutandukanya ibice niterambere
4. Hariho ibimenyetso biranga ibibyimba nizindi ndwara
5. Ibibanza bito bya lncRNA biratandukanye
6. Urukurikirane ruri mukubungabunga, ariko imikorere ifite urwego runaka rwo kubungabunga, nibindi.

02 Ibibazo Bikunze Kubazwa Kubijyanye na LncRNA Guhindura Inyandiko

Bitewe nibirimo bike bya lncRNA mungirangingo, uburebure burebure hamwe nuburyo bwo murwego rwohejuru, RNA ikunze kugira imiterere igoye nka stop loop cyangwa imisatsi, kandi RT-qPCR ikunze guhura nibibazo byo kunanirwa kwandukura cyangwa gukora neza.

03 LncRNA ihindura igisubizo

Mu mategeko ngenderwaho, inzira ya virusi ya RNA ikoresheje inyandiko zabo zinyuranye kugirango zihuze ADN ukoresheje RNA nkicyitegererezo cyitwa reverse transcription.Inzira yo gukoresha virusi ya RNA revers transcriptase kugirango ushushanye cDNA inyandikorugero muri vitro yitwa reverse transcription.Ibyingenzi byingenzi bikubiye muri sisitemu yo kwandukura: RNA inyandikorugero, transcript transcriptase, primers nibindi bice.

1.RNA inyandikorugero

Ingaruka yicyitegererezo cya RNA kumyandikire yinyuma igaragarira muburyo bwayo, ubuziranenge nubunyangamugayo.Iyo hejuru yubuziranenge nubunyangamugayo bya RNA yakuweho, niko imikorere yo kwandukura ihindagurika kandi ibisubizo nyabyo byakurikiranye.RNA yahanaguwe na reagent ikunze gukoreshwa ya RNA ikunze kuba irimo ibisigazwa byinzoga hamwe nu munyu wa guanidine, bigira ingaruka zikomeye zo guhagarika inyandiko zinyuranye.

2.Gusubiramo inyandiko-mvugo

Guhindura inyandiko-mvugo bigira uruhare runini muri sisitemu yose yo kwandukura.Ubushyuhe bukwiye kuri transcript transcript ikoreshwa muri laboratoire nyinshi muri rusange ni 42 ° C.Mubyukuri, gufungura imiterere yo murwego rwohejuru rwa RNA bisaba ubushyuhe bwo hejuru, kandi ibisabwa kuri transcriptase ni byinshi.

3.Primer

Guhindura inyandiko yibanze harimo gene yihariye ya primers, primers random na Oligo dT primers.LncRNAs nyinshi ntabwo zirimo umurizo wa polyA, kandi ni ngombwa guhuza neza primers zidasanzwe na Oligo dT.

4.Ibindi bice

Urebye ko RNA yoroshye cyane gutesha agaciro, kwinjiza ibice muri sisitemu yo kwandukura bishobora kugabanywa, ibyo bikaba bishobora gukumira neza kwinjira kwa RNase kandi bigatuma iterambere ryimyandikire igenda neza.

ForegeneKubona kuva RNA inyandikorugero, guhinduranya inyandiko kuri qPCR

Uherekeza byimazeyo lncRNA yawe

Amatungo yose RNA I.solationKit (hamwe na gDNA yo gukuraho inkingi)

Akagari Cyuzuye RNA I.solationKit (hamwe na gDNA yo gukuraho inkingi)

Lnc-RT IntwariTM I (Hamwe na gDNase) (Super Premix ya synthesis ya cDNA ya mbere kuva lncRNA)

Igihe nyacyo PCR EasyTM-SYBR Icyatsi I.

Aibyiza ofGukuramo RNA:

Igikoresho gikoresha igisekuru cya gatatu RNA ikuramo tekinoroji, nta mpamvu yo gukoresha DNase.

Iminota 11 kuva 96, 24, 12, na 6-isahani yuzuye isahani yimico, gukuramo cyane-gukuramo gDNA-yubusa, isuku-yuzuye, selile yuzuye ya RNA.

Ibyiza bya lncRNA ihinduranya inyandiko:

Sisitemu yo kwandukura sisitemu yakozwe cyane cyane kuri LncRNA kugirango ikureho vuba ADN yanduye.

Sisitemu yo kwandukura sisitemu ifite ubushyuhe bwinshi kandi iracyafite imikorere myiza yo kwandukura kuri 50 ° C.

Inyandikorugero zifite GC nyinshi hamwe nuburyo bugoye bwa kabiri birashobora guhindurwa hamwe nubushobozi buhanitse.

Ibyizaof qPCR:

Gutangira bishyushye Foregene Taq Polymerase ifite ubushobozi bwo kongera imbaraga, ibyiyumvo byiyongera, hamwe nuburyo bwihariye bwo kongera imbaraga

Uburyo bwiza bwa PCR bworoshye buvanze butuma SYBR Icyatsi I ifite sensibilité yo hejuru yo kumenya, kandi ubukana bwa fluorescence bwikubye inshuro 3-5 ibyo bicuruzwa bisa, bishobora guhaza ibyifuzo byubwoko butandukanye bwa fluorescence igerageza.