Ibibazo byaAmatungo yose ya RNA yigunga

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RNA ntabwo yakuweho cyangwa umusaruro wa RNA ni muke

Hariho ibintu byinshi bigira ingaruka kumikorere yo gukira, nka: tissue sample ya RNA ibirimo, uburyo bwo gukora, ingano ya elution, nibindi.

1. Kwiyuhagira urubura cyangwa cryogenic (4 ° C) centrifugation yakozwe mugihe cyo gukora.

Icyifuzo: Kora ku bushyuhe bwicyumba (15-25 ° C) mugihe cyose, ntukarabe hamwe na centrifuge mubushyuhe buke.

2. Kubika icyitegererezo kidakwiye cyangwa igihe cyo kubika urugero.

Icyifuzo: Bika ingero kuri -80 ° C cyangwa uhagarike muri azote yuzuye kandi wirinde gukoresha inshuro nyinshi;gerageza gukoresha tissue nshya cyangwa selile zifite imico yo gukuramo RNA.

3. Lisisi ntangarugero idahagije.

Icyifuzo: Mugihe ingirabuzima fatizo, menya neza ko ingirangingo zahujwe bihagije kandi ko ingirabuzimafatizo zigabanyijemo bihagije kugirango zisobanure irekurwa rya RNA.

4. Umuhanga ntabwo yongeweho neza.

Icyifuzo: Emeza ko ddH idafite RNase₂O yongeweho ibitonyanga hagati yo kweza inkingi ya membrane.

5. Ingano yukuri ya Ethanol yuzuye ntabwo yongewe kuri Buffer RL2 cyangwa Buffer RW2.

Icyifuzo: Kurikiza amabwiriza, ongeramo ingano yukuri ya Ethanol yuzuye kuri Buffer RL2 na Buffer RW2 hanyuma uvange neza mbere yo gukoresha ibikoresho.

6. Dissue sample dosage ntabwo ikwiye.

Icyifuzo: Koresha 10-20 mg ya tissue cyangwa (1-5) × 10⁶selile kuri 500 μl buffer RL1, kuko gukoresha tissue birenze urugero bishobora kugabanya gukuramo RNA.

7. Ingano yo gukuraho idakwiye cyangwa gusiba kutuzuye.

Icyifuzo: Ingano yo gukuraho inkingi yo kweza ni 50-200 μl;niba ingaruka zo gukuraho zidashimishije, birasabwa kongera igihe cyo gushyira ubushyuhe bwicyumba nyuma yo kongeramo ubushyuhe bwa RNase-Ubusa ddH₂O, urugero kuminota 5-10.

8.Inkingi yo kweza ifite ibisigisigi bya Ethanol nyuma yo gukaraba Buffer RW2.

Icyifuzo: Niba hari ibisigisigi bya Ethanol nyuma yo gukaraba Buffer RW2, centrifugation yubusa ya 1min, igihe cyo gukora igituba cyubusa centrifugation gishobora kwiyongera kugeza 2min, cyangwa inkingi yo kweza irashobora gushyirwa mubushyuhe bwicyumba kuminota 5 kugirango ikureho Ethanol isigaye.

RNA isukuye irateshwa agaciro

Ubwiza bwa RNA yatunganijwe bujyanye nibintu nko kubungabunga icyitegererezo, kwanduza RNase, na manipulation, nibindi.

1. Ingero z'imyenda ntizibikwa mugihe.

Icyifuzo: Niba ingero za tissue cyangwa selile zidakoreshejwe mugihe gikwiye nyuma yo kwegeranya, hita ubika neza kuri -80 ° C cyangwa azote yuzuye.Gukuramo RNA, koresha tissue nshya yafashwe cyangwa selile selile igihe cyose bishoboka.

2. Gusubiramo inshuro nyinshi gukonjesha ingero za tissue.

Icyifuzo: Mugihe ubitse ingero za tissue, nibyiza ko ubicamo uduce duto kugirango ubungabunge, hanyuma ukuremo kimwe mubice mugihe ubikoresheje kugirango wirinde gukonjesha inshuro nyinshi no kwangirika kwa RNA.

3. RNase itangizwa cyangwa itambaye uturindantoki twajugunywe, masike, nibindi mugihe cyo gukora.

Icyifuzo: Ubushakashatsi bwo gukuramo RNA bukorwa neza mubyumba bitandukanye bya manipulation ya RNA kandi ameza arasukurwa mbere yubushakashatsi.

Wambare uturindantoki hamwe na masike mugihe cyo kugerageza kugirango ugabanye kwangirika kwa RNA guterwa no kwinjiza RNase.

4. Reagents zandujwe na RNase mugihe cyo kuyikoresha.

Icyifuzo: Simbuza inyamaswa nshya Igiteranyo cya RNA cyo kwigunga kubushakashatsi bujyanye.

5. Imiyoboro ya centrifuge, inama, nibindi bikoreshwa muri manipulation ya RNA byanduye na RNase.

Icyifuzo: Emeza ko imiyoboro ya centrifuge, inama, imiyoboro, nibindi bikoreshwa mugukuramo RNA byose ni RNase-Ubuntu.

RNA yatunganijwe neza igira ingaruka kubushakashatsi bwo hasi

RNA yasukuwe ninkingi yo kweza, niba ion yumunyu, proteyine nini nini cyane bizagira ingaruka kubigeragezo byo hasi, nka: transcription transcription, Blot y'Amajyaruguru nibindi.

1. RNA yakuweho ifite ibisigazwa byumunyu.

Icyifuzo: Emeza ko ingano yukuri ya Ethanol yongewe kuri Buffer RW2 hanyuma ukore inkingi 2 yoza inkingi kumuvuduko wa centrifugal yerekanwe kubikorwa;niba hari ibisigazwa byumunyu ion, usige inkingi yo kweza kuri Buffer RW2 muminota 5 mubushyuhe bwicyumba hanyuma ukore centrifugation kugirango ukureho umwanda mwinshi.

2. Ibisigisigi bya Ethanol muri RNA yatowe.

Icyifuzo: Emeza ko nyuma yo gukaraba buffer RW2, kora ibikorwa byubusa bya centrifugation yubusa kumuvuduko wa centrifugation yerekanwe kubikorwa, ongera igihe cyibikorwa bya centrifugation yubusa kugeza 2 min niba hakiri ibisigisigi bya Ethanol, cyangwa ubirekere mubushyuhe bwicyumba muminota 5 nyuma ya centrifugation yubusa kugirango ukureho ibisigisigi bya Ethanol.

Gutandukanya ubwoko bwa aside nulceic

Ibikoresho bya RNA bya Foregene birashobora kubona RNA yo kwigunga / gukuramo ibintu byose bikurikira: inyama zinyamanswa, ibimera, selile, virusi, amaraso, nibindi.

Nigute nabika ibikoresho

Kubika ubushyuhe bwicyumba amezi 24.