Liphoofolo Kakaretso ea RNA Isolation Kit Kakaretso ea RNA ea ho Ntša le ho Hloekisa Lithako tsa Liphoofolo le Lisele
Tlhaloso ea Kit:
Ka potlako le ka mokhoa o atlehileng o ntša RNA ea boleng bo holimo le ea boleng bo holimo ho tsoa liseleng tse fapaneng tsa liphoofolo.
Ha ho hlokahale hore u tšoenyehe ka ho senyeha ha RNA.Tsamaiso eohle ke RNase-Free
Tlosa DNA ka katleho u sebelisa DNA-Cleaning Column
Tlosa DNA ntle le ho eketsa DNase
E bonolo - ts'ebetso eohle e phethoa ka mocheso oa kamore
Ka potlako-ts'ebetso e ka phethoa ka metsotso e 30
E bolokehile - ha ho na reagent ea tlhaho e sebelisoang
Bohloeki bo phahameng-OD260/280≈1.8-2.1
Litlhaloso tsa Kits
50 Preps, 200 Preps
Sekhahla sena se sebetsaspin kholomo le foromoe entsoeng ke k'hamphani ea rona, e ka ntšang bohloeki bo phahameng le ba boleng bo phahameng ba RNA ho tloha liseleng tse fapa-fapaneng tsa liphoofolo tse nang le bokhoni bo phahameng.E fana ka DNA-Cleaning Column e sebetsang hantle, e ka arolang habonolo le ho bapatsa DNA ea genomic ho tloha ho supernatant le tissue lysate, e bonolo le e bolokang nako;Kholomo ea RNA-feela e ka tlama RNA hantle 'me e ka sebetsoa ka nako e le 'ngoe ka foromo e ikhethang Mehlala e mengata.
Tsamaiso eohle ke RNase-Free, e le hore RNA e nkiloeng e se ke ea senyeha;Buffer RW1, Buffer RW2 buffer system ea ho hlatsoa, e le hore RNA e fumanoeng e se na protheine, DNA, ion, le tšilafalo ea motsoako oa organic.
Likarolo tsa Kit
| Animal Total RNA Isolation Kit | ||
| Likarolo tsa Kit | 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-Free ddH2O | 10ml | 40ml |
| Lekholomo la RNA feela | 50 | 200 |
| DNA-Ho hloekisa Column | 50 | 200 |
| Buka ea Tataiso | 1 sengoathoana | 1 sengoathoana |
Lintlha tsa sehlahisoa
| Sebopeho | Kholomo ea spin | Karolo ea tlhoekiso | Foregene column, reagent |
| Flux | 1-24 mehlala | Nako ka 'ngoe | ~30 mets (24 disampole) |
| Centrifuge | Desk centrifuge | Karohano ea pyrolysis | Karohano ea centrifugal |
| Mohlala | Lisele tsa liphoofolo;sele | Mehlala ea palo | Lithane: 10-20 mg;Sele:(1-5)×106 |
| Elution bophahamo ba modumo | 50-200 μL | Boholo ba molumo oa ho kenya | 850 μL |
Likarolo le melemo
■ Ha ho hlokahale hore u tšoenyehe ka ho senyeha ha RNA;tsamaiso eohle ke RNase-Free
■ Tlosa ka katleho DNA-sebelisa DNA-Cleaning Column
■ Tlosa DNA ntle le ho eketsa DNase
■ Ts'ebetso e bonolo-tsohle e phethoa ka mocheso oa kamore
■ Ts'ebetso e potlakileng e ka phethoa ka mor'a metsotso e 30
■ Sireletsehile-ha ho na reagent ea tlhaho e hlokahalang
■ Bohloeki bo phahameng -OD260/280≈1.8-2.1
Sesebelisoa sa Kit
E loketse ho ntšoa le ho hloekisoa ha RNA e felletseng ho tsoa mefuteng e fapaneng ea lithane tsa liphoofolo tse foreshe kapa tse hatselitsoeng kapa lisele tse holileng.
Lihlahisoa tsa lihlahisoa
■ Lisebelisoa tse tlase: motsoako oa pele oa cDNA, RT-PCR, cloning ea molek'hule, Northern Blot, joalo-joalo.
■ Mehlala: lisele tsa liphoofolo, lisele tse hōlileng tsebong
■ Tekanyetso: Li-tissues 10-20mg, Lisele (2-5)×106
■ Boholo bo tlamang DNA ea kholomo ea tlhoekiso: 80 μg
■ Molumo oa elution: 50-200 μl
Setšoantšo
Animal Total RNA Isolation Kit tšoaroa 20mg
Fresh mouse disampole, nka 5% hloekisitsoeng kakaretso RNA 1% agar
Glycogel electrophoresis
1: Lesapo la 2: Liphio
3: Sebete 4: Pelo
Polokelo le shelf bophelo
Thepa e ka bolokoa likhoeli tse 24 mocheso oa kamore (15-25 ℃) kapa 2-8 ℃ nako e telele.Buffer RL1 e ka bolokoa ho 4 ℃ bakeng sa khoeli ea 1 ka mor'a ho eketsa β- mercaptoethanol (ho ikhethela).
Lingoloa tse qotsitsoeng
1.HAEBA:18.808:Zheng, Q., Qin, F., Luo, R., et al.mRNA-Loaded Lipid-Like Nanoparticles bakeng sa Liver Base Editing ka Ntlafatso ea Moqapi o Bohareng oa Composite.Adv.Mosebetsi.Mater.2021, 31, 2011068.doi:10.1002/adfm.202011068.
2.HAEBA:18.187:He X, Hong W, Yang J, et al.Apoptosis e iketsang ea lisele tsa kalafo ea stem cell e fana ka litlamorao tsa immunomodulatory ka ho lokolloa ha phosphatidylserine.Signal Transduct Target Ther.2021 Jul 14;6(1):270.doi: 10.1038/s41392-021-00688-z.
3.HAEBA:17.97:Dai Z, Liu H, Liao J, et al.Phetoho ea N7-Methylguanosine tRNA e ntlafatsa phetolelo ea oncogenic mRNA hape e khothalletsa tsoelo-pele ea intrahepatic cholangiocarcinoma.Mol Cell.2021 Jul 29:S1097-2765(21)00555-4.doi: 10.1016/j.molcel.2021.07.003.
4.HAEBA:9.225:Cao X, Shu Y, Chen Y, et al.Mettl14-Mediated m6A Phetoho e Thusa ho Tsosolosa Sebete ka ho Boloka Endoplasmic Reticulum Homeostasis.Cell Mol Gastroenterol Hepatol.2021;12(2):633-651.doi: 10.1016/j.jcmgh.2021.04.001.
RNA isoaltion kits bakeng sa mehloli e meng ea mohlalalia fumaneha:
Sele, semela, kokoana-hloko, mali, joalo-joalo.
RNA ha e ntšoe kapa lihlahisoa tsa RNA li tlase
Hangata ho na le lintlha tse fapaneng tse amang ts'ebetso ea ho hlaphoheloa, joalo ka: dikahare tsa RNA tsa sampole, mokhoa oa ts'ebetso, bophahamo ba molumo, jj.
1. Ho hlatsoa leqhoa kapa cryogenic (4 ° C) centrifugation e ne e etsoa nakong ea ts'ebetso.
Keletso: Sebetsa ka mocheso oa kamore (15-25 ° C) ho pholletsa le ts'ebetso eohle, u se ke ua hlatsoa leqhoa le centrifuge ka mocheso o tlaase.
2. Ho boloka sampole e sa nepahalang kapa nako e feteletseng ea ho boloka sampole.
Keletso: Boloka lisampole ho -80 °C kapa u hatsele ka har'a metsi a naetrojene 'me u qobe tšebeliso ea khafetsa ea leqhoa;leka ho sebelisa lisele tse ncha kapa lisele tsa setso bakeng sa ho ntša RNA.
3. Sample e sa lekaneng ea lysis.
Keletso: Ha lesela le kopanya homogenizing, etsa bonnete ba hore lisele li homogenized ka ho lekaneng le hore lisele tsa 'mele li arohane ka ho lekaneng ho hlalosa ho lokolloa ha RNA.
4. Theluent ha e eketsoe ka nepo.
Keletso: Netefatsa hore RNase-Free ddH2O e kenyelletsoa ka tsela e theohang bohareng ba lera la ho hloekisa.
5. Bolumo bo nepahetseng ba absolute ethanol ha boa eketsoa ho Buffer RL2 kapa Buffer RW2.
Keletso: Latela litaelo, eketsa molumo o nepahetseng oa absolute ethanol ho Buffer RL2 le Buffer RW2 'me u kopanye hantle pele u sebelisa khiti.
6. Tekanyetso ea sampole ea lisele ha ea lokela.
Keletso: Sebelisa 10-20 mg ea lisele kapa (1-5) × 106lisele ka 500 μl buffer RL1, kaha tšebeliso e feteletseng ea lisele e ka fella ka ho fokotsoa ha RNA.
7. Molumo o sa nepahalang oa ho hlakisa kapa tlhahiso e sa fellang.
Keletso: Bophahamo ba molumo oa kholomo ea tlhoekiso ke 50-200 μl;haeba phello ea elution e sa khotsofatse, ho khothaletsoa ho eketsa nako ea ho beha mocheso oa kamore ka mor'a ho eketsa RNase-Free ddH2O, mohlala bakeng sa 5-10 min.
8.Kolumo ea tlhoekiso e na le masalla a ethanol ka mor'a hore Buffer RW2 e hlatsoe.
Keletso: Haeba ho na le masala a ethanol ka mor'a ho hlatsoa ha Buffer RW2, tube e se nang letho ea centrifugation bakeng sa 1min, nako ea ts'ebetso ea "tube centrifugation" e se nang letho e ka eketsoa ho 2min, kapa mohala oa tlhoekiso o ka behoa mocheso oa motsoako bakeng sa metsotso e 5 ho tlosa ethanol e setseng ka ho lekaneng.
RNA e hloekisitsoeng e senyehile
Boleng ba RNA e hloekisitsoeng bo amana le lintlha tse kang ho boloka sampole, tšilafalo ea RNase, le ho qhekella, joalo-joalo.
1. Mehlala ea lithane ha e bolokoe ka nako.
Keletso: Haeba lisampole tsa linama kapa lisele li sa sebelisoe ka nako ka mor'a ho bokelloa, hang-hang cryopreserve ho -80 °C kapa naetrojene e metsi.Ho ntša RNA, sebelisa lisele tse sa tsoa nkuoa ha ho khoneha.
2. Ho qhibiliha ka makhetlo-khetlo ha lisampole tsa linama.
Keletso: Ha u boloka lisampole tsa linama, ho molemo ho li khaola likotoana tse nyane bakeng sa ho bolokoa, 'me u tlose e' ngoe ea likotoana ha u li sebelisa ho qoba ho qhibiliha khafetsa ha sampuli le ho senyeha ha RNA.
3. RNase e hlahisoa kapa e sa roala liatlana tse lahloang, limaske, joalo-joalo nakong ea ts'ebetso.
Keletso: Liteko tsa ho ntša RNA li etsoa hantle ka ho fetisisa likamoreng tse arohaneng tsa RNA 'me tafole e hlakoloa pele ho teko.
Apara liatlana le limaske tse lahloang nakong ea liteko ho fokotsa ho senyeha ha RNA ho bakoang ke ho hlahisoa ha RNase.
4. Li-reagents li silafalitsoe ke RNase nakong ea tšebeliso.
Keletso: Kenya sebaka ka Animal Total RNA Isolation Kit bakeng sa liteko tse amanang.
5. Li-tubes tsa centrifuge, malebela, joalo-joalo tse sebelisitsoeng ho fetola RNA li silafalitsoe ke RNase.
Keletso: Netefatsa hore liphaephe tsa centrifuge, malebela, lipeipi, joalo-joalo tse sebelisoang ho RNA ha li na RNase kaofela.
E hloekisitsoeng e fumanoeng RNA e ama liteko tse tlase
RNA e hloekisitsoeng ke kholomo ea tlhoekiso, haeba li-ion tsa letsoai, liprotheine li le ngata haholo li tla ama liteko tse tlase, tse kang: reverse transcription,Northern Blot et al.
1. The elutioned RNA e na le masalla a ione ea letsoai.
Keletso: Netefatsa hore molumo o nepahetseng oa ethanol o kentsoe Buffer RW2 le ho etsa litšepe tse 2 tsa tlhoekiso ka lebelo la centrifugal le bontšitsoeng bakeng sa ts'ebetso;haeba ho na le masala leha e le afe a ion ea letsoai, tlohela kholomo ea tlhoekiso ho Buffer RW2 bakeng sa 5 min mocheso oa kamore 'me u etse centrifugation ho eketsa ho tlosoa ha letsoai.
2. Masala a Ethanol ho RNA e hlahisitsoeng.
Keletso: Etsa bonnete ba hore ka mor'a ho hlatsoa li-buffer RW2, ho etsa ts'ebetso ea centrifugation e se nang letho ka lebelo la centrifugation le bontšitsoeng bakeng sa ts'ebetso, eketsa nako ea ts'ebetso e se nang letho ea tube centrifugation ho 2 min haeba ho ntse ho e-na le masala a ethanol, kapa ue tlohele mocheso oa motsoako bakeng sa metsotso ea 5 ka mor'a hore ho be le letho la tube centrifugation ho eketsa ho tlosoa ha ethanol.
