1. Ziva kunyura kweRNA solution
Absorbance pa280, 320, 230, uye 260 nm inomiririra kukosha kwe nucleic acid, kumashure (solution turbidity), munyu, uye organic matter seprotein, zvichiteerana.Kazhinji chete tarisa OD260/OD280 (Ratio, R).Apo 1.8 ~ 2.0, tinofunga kuti kusvibiswa kweprotein kana zvimwe zvinhu zvehupenyu muRNA zvinogona kushivirira, asi zvinofanira kuonekwa kuti kana Tris inoshandiswa sevhavha yekuona kunyura, kukosha kweR kunogona kuva kwakakura kudarika 2 (kazhinji inofanira kuva <2.2).Apo R <1.8, kusvibiswa kweprotein kana zvimwe zvinhu zvehupenyu mukugadzirisa zvinonyanya kuoneka, uye kuguma kweRNA kunogona kugadziriswa maererano nezvinodiwa.Kana R> 2.2, zvinoreva kuti RNA yakave yakaitwa hydrolyzed kuita imwechete nucleic acid.
2.Electrophoretic pattern yeRNA
Kazhinji, denaturing gel inoshandiswa kuRNA electrophoresis, asi kana iri yekuona mhando yeRNA, denaturing gel haifanirwe, uye yakajairika agarose gel inogona kushandiswa.Chinangwa che electrophoresis ndechekuona kuvimbika kwe28S uye 18S mabhendi uye chiyero chavo, kana kuvimbika kwemRNA smear.Kazhinji, kana mabhendi e28S uye 18S akajeka, akajeka, uye akapinza (zvichireva kumicheto yemabhandi akajeka), uye kupenya kwe28S kwakapetwa kaviri kune 18S band, tinofunga kuti kunaka kweRNA kwakanaka.
Idzi dziri pamusoro ndidzo nzira mbiri dzatinowanzo shandisa, asi hapana panzira mbiri idzi inogona kutiudza zvakajeka kana paine RNase yasara mumhinduro yeRNA.Kana pane chidimbu chidiki cheRNase mumhinduro, zvakaoma kuti isu tizvione nenzira iri pamusoro, asi yakawanda yeanotevera maitiro enzymatic inoitwa pamusoro pe37 degrees uye kwenguva yakareba.Nenzira iyi, kana paine shoma shoma yeRNase muRNA mhinduro, ipapo pachava nenzvimbo yakakodzera kwazvo uye nguva yekuita basa ravo mukuedza kunotevera, uye chokwadi kuedza kuchatonhora panguva ino.Pazasi tinosuma nzira inogona kusimbisa kana paine yasara RNase muRNA mhinduro.
3. Muedzo wekuchengetedza kupisa
Zvinoenderana nesample concentration, dhirowa maviri 1000 ng RNA kubva mumhinduro yeRNA woiwedzera kune 0.5 ml centrifuge chubhu, woiwedzera nepH 7.0 Tris buffer kusvika kuhuwandu hwegumi ul, wobva wasunga chivharo chechubhu.Isa chimwe chazvo mubhati yemvura yekushisa nguva dzose pa 70 ° C uye uichengete ichidziya kwe1 h.Chimwe chikamu chakachengetwa mu -20 ° C firiji ye1 h.Kana nguva yasvika, bvisa masampuli maviri e electrophoresis.Mushure mokunge electrophoresis yapera, enzanisa mabhandi e electrophoretic ezviviri.Kana mabhanhire ezviviri izvi achienderana kana kuti asina mutsauko wakakosha (zvechokwadi, mabhendi avo anosanganawo nemamiriro emunzira 2), zvinoreva kuti hapana yakasara RNase kusvibiswa muRNA mhinduro, uye mhando yeRNA yakanaka kwazvo.Pane zvinopesana, kana sampu yakanyungudutswa pa70 ° C inoratidza kuparara kuri pachena, inoratidza kuti kune kusvibiswa kweRNase muRNA mhinduro.
2 Nzira dzekuyedza uye matekiniki eRNA kuburitsa
Matambudziko atinowanzosangana nawo pakuchera RNA ndeaya: (1) Goho reRNA rakaderera;(2) RNA ine kushatisa munyu kwakakomba;(3) RNA ine zvakakomba organic solvent kusvibiswa;(4) kuderedzwa kwemuenzaniso uye mamwe matambudziko
1. Inowanzoshandiswa zvachose RNA kutorwa reagents
Nzira ye guanidine isothiocyanate uye nzira yeTrizol ndiyo nzira dzinonyanya kushandiswa pakubvisa RNA yose kubva munyama yemhuka uye masero emhuka.Inonyanya kukodzera masampuli maduku uye matishu anonyanya kuoma kubvisa, zvakadai sekubviswa kweRNA yose kubva paganda retsuro uye mhuka yekubatanidza mhuka;uyezve, Trizol, seyakajairwa-chinangwa lysis reagent, inogona zvakare kushandiswa kudhirowa kwemiti yezvirimwa, mabhakitiriya, fungi uye mamwe matissue.Kune matishu ezvirimwa ane polysaccharides uye polyphenols, senge camellia oleifera, tii mashizha, rapeseed, nezvimwewo, iyo CTAB nzira inogona zvakare kushandiswa kubvisa yakazara RNA.
Senzira yakajairika, iyo mbiri-column nzira inofarirwawo zvakanyanya nekuda kweiyo yakajairika tembiricha kushanda, hapana chikonzero chekuwedzera RNase, uye chengetedzo-hapana chloroform, phenols uye mamwe organic reagents ekubvisa.(zvinokurudzirwa zvigadzirwa )
2. Kubviswa kweRNA yose kubva munyama yemhuka
(1) Edza kusarudza matishu matsva, kana asina kutsva (zvichida mukati memwedzi mitatu - 80 ℃ firiji kana kuomeswa nenitrogen yemvura. Pakucheka tishu, usacheka zvakananga pane tembiricha yekamuri, iva nechokwadi chekuisa pabhokisi rechando, edza kudzivirira kutonhora kudzokororwa nekunyunguduka.
(2) Shandisa zvigero zvakachena uye ma tweezers kucheka chidimbu chidiki chematishu, edza kucheka chikamu chepakati chetishu paunenge uchicheka samuenzaniso, kana tanga wacheka chimedu chihombe chetishu kubva nepakati, wobva wacheka sample panzvimbo patsva.Iyo matishu yakabviswa inofanirwa kuve yakanyungudutswa zvizere, isa shredded tishu mu EP chubhu isina RNase, wedzera iyo lysate, iyo shredded tishu inofanirwa kuratidzwa zvizere kune lysate, uye gadzirira homogenization.
(3) Pazvinyama zvakajairika, sarudza mung bhinzi-yakakura matissue (30-60 mg) yehomogenization.Kana matishu ane mapuroteni akawanda, mafuta, kana dense fibrous tissues sechiropa, zvakakodzera kuwedzera kana kuderedza uwandu hwekucheka nyama (optional) Sarudza 10 ~ 20 mg).
(4) Kana hove tsandanyama, shrimp nyama, jellyfish uye mamwe matishu ane yakakwira yemvura akaburitswa, vhoriyamu yemuenzaniso inofanira kuwedzerwa zvakakodzera (inokurudzirwa 100-200 mg).
(5) Kana mamiriro ezvinhu achibvumira, mhuka yemhuka inogona kutorwa zvakananga mushure mekuita homogenized ne-high-passage tissue homogenizer, kana pasina michina yakadaro.
(6) Iyo RNA yakawanikwa mushure mekubviswa kwekupedzisira inofanira kuiswa pabhokisi rechando nekukurumidza kuderedza kuderedzwa kweRNA.
3. Mhuka sero RNA kutorwa
(1) Masero ekusimudzwa: centrifuge zvakananga uye kurasa svikiro, geza nePBS isina kuberekwa kwe1-2 nguva, wozomisa nehuwandu hwakakodzera hwePBS, uye wobva wawedzera lysate ye lysis.Usawedzera lysate zvakananga kune masero anonaya mushure mekurasa zvachose mvura.Izvi zvinozoita kuti histone package inoburitswa mushure meiyo lysed maseru ari pakunze kwe layer kuti inamate kune kunze kwemaseru anonaya, nekudaro ichidzikamisa kusangana kwemaseru mukati me pellet ne lysate., zvichikonzera kusakwana kwesero lysis uye kuderedza RNA goho.
(2) Masero ari semi-adherent kana asinganyatso kuomerera: Mushure mokurasa svikiro, gezai nePBS kwe1-2 nguva, wobva watora zvakananga huwandu hwakakodzera hwePBS uye ufuridze ndiro yetsika nepipette kana pfuti kuti ivhuvhute masero, uye woaendesa kumasero asina RNA.Wedzera lysate kune EP chubhu ye enzyme kuti ibvise.
(3) Masero anonamatira: anoda kugayiwa netrypsin kutanga, wozounganidzwa muRNase-isina EP machubhu, centrifuged kubvisa iyo supernatant, yakashambidzwa 1-2 nguva nePBS kubvisa yakawandisa trypsin, uye kudzoreredzwa nehuwandu hwakakodzera hwePBS Zvadaro enda kudanho rekubvisa.
4. Plant RNA kutorwa
Miti yemiti yakapfuma mu phenolic compounds, kana hupfumi mumapolysaccharides, kana ine mamwe mametabolites echipiri asingazivikanwe, kana ane basa rakakwirira reRNase.Izvi zvinhu zvakabatanidzwa zvakasimba neRNA mushure mesero lysis kuti iite insoluble complexes kana colloidal precipitates, iyo yakaoma kubvisa.Naizvozvo, kana isu tichibvisa chirimwa matishu, isu tinofanirwa kusarudza kititi chezvirimwa.Iyo lysate mukiti inogona kugadzirisa zvinobudirira matambudziko e nyore oxidation ye polyphenols uye kupatsanurwa kwepolysaccharide makomisheni uye nucleic acids.
(Nezve polysaccharide polyphenol chirimwa RNA kudhirowa, zvinokurudzirwa zvigadzirwa:
(1) Pepa, pulp, mhodzi, mashizha, nezvimwe zvechirimwa zvinofanira kunyatsokuyiwa mudhaka.Munguva yekukuya, mvura ine nitrogen inofanira kuzadzikiswa nenguva kuti isanyungudutse sampuli.Sampu yepasi inofanira kukurumidza kuwedzerwa kune lysate uye kuzununguswa kudzivirira RNA kuora.
(2) Kune sampuli dzakapfuma-fiber dzakadai semupunga nemashizha egorosi, huwandu hwekuchera hunofanira kuderedzwa zvakakodzera, kana zvisina kudaro kukuya kwenyama uye lysis hakuzove kwakakwana, zvichiita kuti pave negoho rakaderera reRNA yakabviswa.
(3) Kune matishu ezvirimwa ane yakakwira yemvura, senge pomegranate muchero, muchero wewatermelon, peach muchero, nezvimwewo, saizi yemuenzaniso inofanira kuwedzerwa zvakakodzera (100-200 mg inosarudzwa).
(4) Matishu ekudyara, akadai semashizha echirimwa, marhizomes, michero yakaoma uye zvimwe zvinhu zvinowanzokurudzirwa kushandisa nitrogen yemvura kunyatsogadzira zvimisikidzo mudhaka, wozoenderera mberi nedanho rekubvisa.Yakajairwa tishu homogenizers inogona kusashanda mukuita homogenizing matishu ezvirimwa, uye kazhinji haina kukurudzirwa.
5. Chengetedzo yeRNA kutorwa
(1) Matishu esampuli anofanira kunge ari matsva sezvinobvira kudzivirira kudzokororwa nechando uye kunyunguduka.
(2) Nyama yacho inofanira kunge yakanyatsokuyiwa painobviswa, uye uwandu hwenyama hunofanira kuva hushoma, ndoda kuwanda.
(3) Yakakwana incubation nguva inofanirwa kupihwa mushure mekuwedzera lysate kuti inyatso lyse sampu.
(4) Paunenge uchishandisa nzira yeTrizol yekubvisa, musimboti wekutora iyo supernatant mushure me stratification "inofarira kufemesa zvishoma pane inhale yakawanda", uye haifanirwe kuburitsa kune yepakati layer, zvikasadaro zvinokonzeresa yakakomba genomic DNA kusvibiswa.
(5) Pakugeza, mvura yekuwachisa inofanira kupinda mukati memadziro echubhu kuti inyatsogezwa.
(6) Kune nzira yekubvisa mbiru, pamusoro pekubvisa mbiru mushure mekugeza, iyo adsorption mbiru inofanirawo kuiswa mubhenji rakachena kwazvo uye rinofuridzwa kwemaminetsi 5-10 kuti inyungurudze zvizere organic solvent kusvika pakuoma.
(7) Panguva yekupedzisira yenzira yekoramu, mushure mekuwedzera DEPC mvura, inofanira kuvharirwa kwemaminitsi 3-5, kana mvura yeDEPC inofanira kupisa kusvika 60 ° C mberi kuti iwedzere muchero we elution.Muchinyakare cheTrizol cleavage uye isopropanol precipitation method, RNA yekupedzisira inonyungudutswa mumvura yeDEPC, saka nguva yakakodzera inofanira kupiwa yekuparara, uye pasi pe centrifuge tube inofanira kuramba ichiputirwa nepipette tip.
3 Three Zvikonzero uye zvigadziriso zvekudzika kweRNA kusungwa / kushata kwemhando
1. Goho rakaderera
Muenzaniso wakabudiswa wakanyanya kuderera, mari yose haina kukwana, kana sampuli yakabudiswa yakawandisa uye lysis haina kukwana;iyo mhuka kana masero ehutano hwakakodzera hunofanira kushandiswa kubviswa, kusati kwaitwa kurapwa kwemuenzaniso kunofanira kuitwa zvakanaka, uye lysis inofanira kukwana.
2. Zvisaririra zveGenome
Kana uchibvisa neTrizol nzira, kana iyo supernatant ichiyamwa muchikamu chepakati mushure mekuisa, kusvibiswa kwakakomba kwegenome kuchakonzerwa;kunyanya kutarisirwa kunofanira kutorwa paunenge uchiisa kudzivirira kusveta mukati mechikamu chepakati.Kana iyo nzira yekoramu yakashandiswa kudhirowa, kit ine DNase Ini ndinogona kusarudzwa kuti ibvise.Iyo nucleic acid yakashambadzirwa pa membrane inogayiwa zvakananga neDNase I, inogona kuderedza zvakanyanya masara eDNA.
3. RNA kushatisa
Inogona kunge iri kuderedzwa kweiyo yakabviswa sampuli pachayo, kana kusvibiswa kunokonzerwa panguva yekubvisa;sezvingabvira, sampuli nyowani dzinofanirwa kushandiswa paRNA kudhirowa, uye sampuli dzakaunganidzwa dzinofanira kuchengetwa mumvura ine nitrogen kana -80 ° C mufiriji nenguva, uye kudzokororwa nechando uye kunyungudutswa kunofanira kudziviswa.RNase/DNase matipi emahara, machubhu ecentrifuge uye zvimwe zvinhu zvinofanirwa kushandiswa muRNA yekubvisa maitiro.Nzira yekubvisa inofanira kunge ichikurumidza sezvinobvira.Iyo RNA yakabviswa inofanira kuiswa pabhokisi rechando uye kuchengetwa pa -80 munguva.Kana iyo RNA yakabviswa ichida kuonekwa ne gel electrophoresis, electrophoresis inofanira kuitwa pakarepo mushure mekubviswa, uye electrophoresis buffer inofanira kuchinjwa neyakagadzirirwa.
4. Munyu uye organic solvent residues
Iyo yekubvisa reagents ine phenol uye guanidine munyu, uye mhinduro yekugeza ine ethanol.Panguva yekuchera, iyo lysate haina kubatwa zvachose uye yakaraswa, uye mushonga wekugeza hauna kunyatsoomeswa.Residual munyu uye organic solvents zvinokuvadza kune inotevera reverse transcription uye PCR.Different degrees of inhibition, saka iyo lysate yenyama inofanira kubviswa zvakakwana panguva yekubvisa, uye kushambidza kunofanira kukwana kuitira kuti masvingo akapoteredza echubhu anogona kushambidzwa.Mukuwedzera, iyo chubhu inodururwa uye inoputika inhanho inodiwa, iyo inowedzera kuderedza zvakasara zvehupenyu.
Kuti uwane rumwe ruzivo nezve RNA kubviswa, ndapota tevera webhusaiti yedu:
www.foreivd.com kuti uwane rumwe ruzivo.
Nguva yekutumira: Zvita-01-2022
