Amatungo Yuzuye RNA Yigunga Igikoresho Cyuzuye RNA Gukuramo no Kweza Ibikoresho Byinyamanswa & Akagari
Ibisobanuro:
Byihuse kandi neza gukuramo RNA-yuzuye kandi yuzuye-yuzuye muri RNA inyama zinyamaswa zitandukanye.
Ntibikenewe ko uhangayikishwa no kwangirika kwa RNA.Sisitemu yose ni RNase-Yubusa
Kuraho neza ADN ukoresheje Inkingi-Yeza
Kuraho ADN utongeyeho DNase
Byoroshye - ibikorwa byose birangirira ku bushyuhe bwicyumba
Byihuse - ibikorwa birashobora kurangira muminota 30
Umutekano - nta reagent ikoreshwa
Isuku ryinshi - OD260 / 280≈1.8-2.1
Kits Ibisobanuro
50 Imyiteguro, 200 Imyiteguro
Iki gikoresho gikoreshakuzenguruka inkingi na formulayatejwe imbere nisosiyete yacu, ishobora gukuramo isuku nini kandi yujuje ubuziranenge RNA mu nyama zinyamanswa zitandukanye kandi ikora neza.Bitanga inkingi ikora neza ya ADN-isukura, ishobora gutandukanya byoroshye kandi ikanatanga ADN ya genomic na lysate ndengakamere na tissue, byoroshye kandi bitwara igihe;Inkingi ya RNA yonyine irashobora guhuza RNA neza kandi irashobora gutunganyirizwa icyarimwe hamwe na formula idasanzwe Ingero nyinshi.
Sisitemu yose ni RNase-Yubusa, kugirango RNA yakuweho itangirika;Buffer RW1, Buffer RW2 sisitemu yo gukaraba, kugirango RNA yabonetse idafite proteyine, ADN, ion, hamwe n’umwanda wanduye.
Ibigize ibikoresho
| Inyamaswa Igiteranyo Cyuzuye RNA | ||
| Ibigize ibikoresho | RE-03011 | RE-03014 |
| 50 T. | 200 T. | |
| Buffer RL1 * | 25ml | 100ml |
| Buffer RL2 | 15ml | 60ml |
| Buffer RW1 * | 25ml | 100ml |
| Buffer RW2 | 24ml | 96ml |
| RNase Yubusa ddH2O | 10ml | 40ml |
| Inkingi ya RNA gusa | 50 | 200 |
| Inkingi ya ADN | 50 | 200 |
| Igitabo gikubiyemo amabwiriza | Igice 1 | Igice 1 |
Amakuru y'ibicuruzwa
| Imiterere | Inkingi | Ibigize kweza | Inkingi ya Foregene, reagent |
| Flux | Ingero 1-24 | Igihe cyo kwitegura | ~ 30 min (ingero 24) |
| Centrifuge | Ameza centrifuge | Gutandukanya Pyrolysis | Gutandukana hagati |
| Icyitegererezo | Inyama z'inyamaswa;selire | Ingero zingana | Tissue: 10-20 mg;Akagari: (1-5) × 106 |
| Ingano yo gukuraho | 50-200 μL | Umubare ntarengwa wo gupakira | 850 μL |
Ibiranga ibyiza
■ Ntibikenewe ko uhangayikishwa no kwangirika kwa RNA;sisitemu yose ni RNase-Ubuntu
Kuraho neza ADN ukoresheje ADN-Isukura Inkingi
Kuraho ADN utongeyeho DNase
Ibikorwa byoroshye-byose birangirira ku bushyuhe bwicyumba
■ Kwihuta -ibikorwa birashobora kurangira muminota 30
■ Umutekano-nta reagent ukenewe
Pure Isuku ryinshi -OD260 / 280≈1.8-2.1
Porogaramu
Birakwiriye gukuramo no kweza RNA yose mubice bitandukanye byinyamanswa nshya cyangwa bikonje cyangwa ingirabuzimafatizo.
Ibipimo byibicuruzwa
Applices Porogaramu yo hasi: synthesis ya mbere ya cDNA, RT-PCR, clone ya molekile, Blot y'Amajyaruguru, nibindi.
Ingero: inyama zinyamanswa, ingirabuzimafatizo
Ingano: Tissue 10-20mg, Ingirabuzimafatizo (2-5) × 106
Ubushobozi ntarengwa bwa ADN bwo guhuza inkingi yo kweza: 80 μg
Volume Ingano yo gukuraho: 50-200 μl
Igishushanyo
Amatungo Yuzuye RNA Yigunze Igikoresho cyavuwe 20mg
Icyitegererezo cyimbeba, fata 5% isukuye yose RNA 1% agar
Glycogel electrophoreis
1: Impyiko 2: Impyiko
3: Umwijima 4: Umutima
Ububiko nubuzima bwiza
Igikoresho gishobora kubikwa amezi 24 mubushyuhe bwicyumba (15-25 ℃) cyangwa 2-8 ℃ mugihe kirekire.Buffer RL1 irashobora kubikwa kuri 4 ℃ ukwezi 1 nyuma yo kongeramo merc mercaptoethanol (bidashoboka).
Ingingo zavuzwe
1.NIBA: 18.808:Zheng, Q., Qin, F., Luo, R., n'abandi.mRNA-Yuzuye Lipid-Nka Nanoparticles yo Guhindura Umwijima Binyuze muri Optimisation yo Hagati Igishushanyo mbonera.Umujyanama.Imikorere.Ibikoresho.2021, 31, 2011068.doi: 10.1002 / adfm.202011068.
2.NIBA: 18.187:We X, Hong W, Yang J, n'abandi.Apoptose ya selile mu buryo bwo kuvura ingirangingo ngengabuzima itegura igira ingaruka zo gukingira indwara binyuze mu kurekura fosifatiqueylserine.Ikimenyetso cyo Gutwara Intego Intego.2021 Nyakanga 14; 6 (1): 270.doi: 10.1038 / s41392-021-00688-z.
3.NIBA: 17.97: Dai Z, Liu H, Liao J, n'abandi.N7-Methylguanosine tRNA guhindura byongera ubusobanuro bwa oncogenic mRNA kandi biteza imbere cholangiocarcinoma intrahepatic.Akagari ka Mol.2021 Nyakanga 29: S1097-2765 (21) 00555-4.doi: 10.1016 / j.molcel.2021.07.003.
4.NIBA: 9.225: Cao X, Shu Y, Chen Y, n'abandi.Mettl14-Yunganirwa na m6A Guhindura byorohereza kuvugurura umwijima ukomeza Endoplasmic Reticulum Homeostasis.Akagari Mol Gastroenterol Hepatol.2021; 12 (2): 633-651.doi: 10.1016 / j.jcmgh.2021.04.001.
RNA kwigunga ibikoresho bya izindi ntangarugerozirahari:
Akagari, ibimera, virusi, amaraso, nibindi
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.
