1. Thola ukumunca kwesisombululo se-RNA
I-Absorbance ku-280, 320, 230, kanye ne-260 nm imele amanani e-nucleic acid, isizinda (isixazululo se-turbidity), ukugxila kukasawoti, kanye ne-organic matter efana neprotheni, ngokulandelanayo.Ngokuvamile bheka kuphela i-OD260/OD280 (Isilinganiselo, R).Uma u-1.8~2.0, sicabanga ukuthi ukungcoliswa kwephrotheni noma enye into ephilayo ku-RNA kungabekezelelwa, kodwa kufanele kuqashelwe ukuthi uma i-Tris isetshenziswa njengebhafa ukuze kutholwe ukumunca, inani lika-R lingase libe likhulu kuno-2 (ngokuvamile kufanele libe ngu-<2.2).Lapho i-R<1.8, ukungcoliswa kwamaprotheni noma ezinye izinto eziphilayo esixazululweni kubonakala ngokucacile, futhi isiphetho se-RNA singanqunywa ngokuvumelana nezidingo.Uma i-R>2.2, kusho ukuthi i-RNA ifakwe i-hydrolyzed ibe yi-nucleic acid eyodwa.
2.Iphethini ye-Electrophoretic ye-RNA
Ngokuvamile, ijeli ye-denaturing isetshenziselwa i-RNA electrophoresis, kodwa uma kuphela ukuthola ikhwalithi ye-RNA, ijeli ye-denaturing ayidingeki, futhi ijeli ye-agarose evamile ingasetshenziswa.Injongo ye-electrophoresis ukuthola ubuqotho bamabhendi angu-28S kanye ne-18S kanye nesilinganiso sawo, noma ubuqotho be-mRNA smear.Ngokuvamile, uma amabhendi e-28S kanye ne-18S egqamile, ecacile, futhi ecijile (ebhekisela emaphethelweni amabhendi acacile), futhi ukukhanya kwe-28S kungaphezu kokuphindwe kabili kokwebhendi ye-18S, sibheka ikhwalithi ye-RNA njengenhle.
Okungenhla yizindlela ezimbili esivame ukuzisebenzisa, kodwa ayikho kulezi zindlela ezimbili engasitshela ngokucacile ukuthi ingabe ikhona yini i-RNase eyinsalela kusixazululo se-RNA.Uma kukhona inani elincane kakhulu le-RNase esixazululo, kunzima ngathi ukukubona ngale ndlela engenhla, kodwa ukusabela okuningi okulandelayo kwe-enzymatic kwenziwa ngaphezu kwama-degrees angu-37 futhi isikhathi eside.Ngale ndlela, uma kunenani elincane kakhulu le-RNase kusixazululo se-RNA, khona-ke kuyoba khona indawo efanelekile kakhulu nesikhathi sokudlala indima yabo ekuhlolweni okulandelayo, futhi ngokuqinisekile ukuhlolwa kuzobanda ngalesi sikhathi.Ngezansi sethula indlela engaqinisekisa ukuthi ingabe ikhona yini i-RNase eyinsalela kusixazululo se-RNA.
3. Ukuhlolwa kokugcina ukushisa
Ngokuhambisana nesampula yokugxilisa ingqondo, dweba i-1000 ng RNA emibili kusixazululo se-RNA bese uyifaka eshubhuni ye-centrifuge engu-0.5 ml, bese uyifakela nge-pH 7.0 Tris buffer ibe isamba sevolumu engu-10 ul, bese uvala isivalo seshubhu.Faka enye yazo endaweni yokugeza yamanzi okushisa okungaguquki ku-70 ° C futhi uyigcine ifudumele ihora elingu-1.Enye ingxenye igcinwe ku -20 ° C esiqandisini isikhathi esingu-1.Uma isikhathi sesiphelile, susa amasampula amabili e-electrophoresis.Ngemuva kokuthi i-electrophoresis isiqediwe, qhathanisa ama-electrophoretic bands kokubili.Uma amabhendi walokhu okubili ehambisana noma engenawo umehluko obalulekile (yebo, amabhendi awo nawo ahlangabezana nemibandela endleleni 2), kusho ukuthi akukho ukungcoliswa kwe-RNase okusele kusixazululo se-RNA, futhi ikhwalithi ye-RNA yinhle kakhulu.Ngokuphambene nalokho, uma isampula efakwe ku-70°C ibonisa ukuwohloka okusobala, kubonisa ukuthi kukhona ukungcoliswa kwe-RNase kusixazululo se-RNA.
2 Izindlela namasu okuhlola okukhishwa kwe-RNA
Izinkinga esivame ukuhlangana nazo lapho sikhipha i-RNA yilezi: (1) Isivuno se-RNA siphansi;(2) I-RNA inokungcoliswa kukasawoti okungathi sína;(3) I-RNA inokungcola okungathi sína kwezinto ezincibilikayo;(4) ukucekelwa phansi kwesampula nezinye izinkinga
1. Ama-reagents okukhipha i-RNA asetshenziswa ngokuvamile
Indlela ye-guanidine isothiocyanate kanye nendlela ye-Trizol yizindlela ezisetshenziswa kakhulu zokukhipha i-RNA ephelele ezicutshini zezilwane namaseli ezilwane.Ifaneleka ngokukhethekile amasampula amancane nezicubu okunzima kakhulu ukuzikhipha, njengokukhishwa kwesamba se-RNA esikhumbeni sikanogwaja kanye nezicubu ezixhumene nezilwane;ngaphezu kwalokho, i-Trizol, njenge-reagent yenhloso evamile ye-lysis, ingabuye isetshenziselwe ukukhipha izicubu zezitshalo, amagciwane, isikhunta nezinye izicubu.Kuzicubu zezitshalo eziqukethe ama-polysaccharides nama-polyphenols, njenge-camellia oleifera, amaqabunga etiye, imbewu ye-rapese, njll., indlela ye-CTAB ingasetshenziswa futhi ukukhipha i-RNA ephelele.
Njengendlela evamile, indlela yekholomu ephindwe kabili ibuye idume kakhulu ngenxa yokusebenza kwayo okujwayelekile kwezinga lokushisa, asikho isidingo sokwengeza i-RNase, nokuphepha-akukho chloroform, ama-phenol namanye ama-reagents e-organic ukuze akhishwe.(imikhiqizo enconyiwe )
2. Ukukhishwa kwesamba se-RNA ezicutshini zezilwane
(1) Zama ukukhetha izicubu ezintsha, uma zingentsha (okungcono zingakapheli izinyanga ezintathu - 80 ℃ isiqandisi noma iqandiswe inayithrojeni ewuketshezi. Lapho usika izicubu, ungasiki ngokuqondile endaweni yokushisa yasekamelweni, qiniseka ukuthi Uyibeka ebhokisini leqhwa, zama ukugwema ukubanda okuphindaphindiwe nokuncibilika.
(2) Sebenzisa isikelo esihlanzekile nama-tweezers ukuze usike ucezu oluncane lwethishu, zama ukusika ingxenye emaphakathi yethishu lapho usika isampula, noma uqale usike ucezu olukhulu lwethishu phakathi nendawo, bese usika isampula endaweni entsha yokusika.Izicubu ezisusiwe kufanele zihlakazwe ngokugcwele, zifake izicubu ezichotshoziwe zibe yithubhu ye-EP ngaphandle kwe-RNase, engeza i-lysate, izicubu ezincibilikisiwe kufanele zivezwe ngokugcwele ku-lysate, futhi zilungiselele i-homogenization.
(3) Ngezicubu ezijwayelekile, khetha izicubu ezilingana nobhontshisi we-mung (30-60 mg) ukuze uthole i-homogenization.Uma izicubu ziqukethe inani elikhulu lamaprotheni, amafutha, noma izicubu eziminyene ezinjengesibindi, khulisa ngokufanelekile noma unciphise inani lezicubu ezisikiwe (uma uthanda) Khetha u-10~20 mg).
(4) Uma imisipha yenhlanzi, inyama yezimfanzi, i-jellyfish nezinye izicubu ezinamanzi amaningi zikhishwa, umthamo wesampula kufanele ukhuliswe ngokufanelekile (kunconywe u-100-200 mg).
(5) Uma izimo zivuma, isicubu sesilwane singakhishwa ngokuqondile ngemva kokuhlanganiswa ne-homogenizer yethishu ephezulu, uma ingekho leyo mpahla.
(6) I-RNA etholwe ngemva kokukhishwa kokugcina kufanele ibekwe ebhokisini leqhwa ngokushesha ukuze kuncishiswe ukucekelwa phansi kwe-RNA.
3. Animal cell RNA isizinda
(1) Amaseli amisiwe: i-centrifuge ngokuqondile futhi ulahle okuphakathi, geza nge-PBS oyinyumba izikhathi ezingu-1-2, bese umisa ngenani elifanele le-PBS, bese wengeza i-lysate ye-lysis.Ungafaki i-lysate ngqo kumaseli anciphile ngemva kokulahla ngokuphelele uketshezi.Lokhu kuzodala ukuthi iphakethe le-histone elikhishwe ngemuva kokuthi amaseli a-lysed asongqimbeni olungaphandle anamathele ngaphandle kwamaseli anciphile, ngaleyo ndlela anciphise ukuthintana kwamaseli ngaphakathi kwe-pellet nge-lysate., okubangela ukuguqulwa kwamaseli okungaphelele nokunciphisa isivuno se-RNA.
(2) Amangqamuzana anamathela kancane noma angabambeleli ngokuqinile: Ngemva kokulahla okuphakathi, geza nge-PBS izikhathi ezingu-1-2, bese udonsa ngokuqondile inani elifanele le-PBS futhi ushaye indishi yesiko nge-pipette noma isibhamu ukuze uqhume amaseli, futhi uwadlulisele kumaseli angenayo i-RNA.Faka i-lysate kushubhu ye-EP ye-enzyme ukuze ikhishwe.
(3) Amaseli anamathelayo: adinga ukugaywa nge-trypsin kuqala, bese eqoqwa kumashubhu e-EP angenayo i-RNase, afakwe i-centrifuged ukuze akhiphe amandla angaphezu kwavamile, agezwe izikhathi ezingu-1-2 nge-PBS ukuze akhiphe i-trypsin eyeqile, futhi aphinde amiswe ngenani elifanele le-PBS Bese uqhubekela esinyathelweni sokukhipha.
4. Isizinda se-RNA yezitshalo
Izicubu zezitshalo zicebile ngamakhompiyutha e-phenolic, noma acebile ngama-polysaccharides, noma aqukethe ama-metabolite esibili angaziwa, noma anomsebenzi ophezulu we-RNase.Lezi zinto zihlanganiswe ngokuqinile ne-RNA ngemuva kwe-cell lysis ukuze zenze izingxube ezingancibiliki noma i-colloidal precipitates, okunzima ukuyisusa.Ngakho-ke, lapho sikhipha izicubu zezitshalo, sidinga ukukhetha ikhithi yezitshalo.I-lysate ku-kit ingaxazulula ngokuphumelelayo izinkinga ze-oxidation elula ye-polyphenols kanye nokuhlukaniswa kwama-polysaccharide compounds nama-nucleic acid.
(Okwe-polysaccharide polyphenol plant RNA isizinda, imikhiqizo enconyiwe:
(1) Ikhasi, uginindela, imbewu, amaqabunga, njll. zesitshalo kufanele zigaywe ngokuphelele odakeni.Ngesikhathi sokugaya, i-nitrogen ewuketshezi kufanele igcwaliswe ngesikhathi ukuze kugwenywe ukuncibilika kwesampula.Isampula yomhlabathi kufanele yengezwe ngokushesha ku-lysate futhi inyakaziswe ukuze kugwenywe ukuwohloka kwe-RNA.
(2) Kumasampula ane-fiber acebile njengerayisi namaqabunga akolweni, inani lokukhishwa kufanele lincishiswe ngokufanelekile, ngaphandle kwalokho ukugaya izicubu kanye ne-lysis ngeke kuphelele, okuholela ekuvuthweni okuphansi kwe-RNA ekhishiwe.
(3) Ngezicubu zezitshalo ezinamanzi amaningi, njengesithelo sehalananda, isithelo sekhabe, isithelo sepentshisi, njll., usayizi wesampula kufanele ukhuliswe ngokufanelekile (100-200 mg uyazikhethela).
(4) Izicubu zezitshalo, njengamaqabunga ezitshalo, ama-rhizomes, izithelo eziqinile nezinye izinto ngokuvamile zituswa ukuba zisebenzise i-nitrogen ewuketshezi ukuze zifake kahle izithako odakeni, bese uqhubekela esinyathelweni sokukhipha.Ama-tissue homogenizers ajwayelekile angase angasebenzi kahle ekuhlanganiseni izicubu zezitshalo, futhi ngokuvamile awanconywa.
5. Izinyathelo zokuphepha zokukhishwa kwe-RNA
(1) Amasampula ezicubu kufanele abe masha ngangokunokwenzeka ukuze agweme ukuqhwanda okuphindaphindiwe nokuncibilika.
(2) Izicubu kufanele zigaywe ngokugcwele lapho zikhishwa, futhi inani lezicubu akufanele libe lincane kakhulu, ingasaphathwa eyeqile.
(3) Isikhathi esanele sokufukamela kufanele sinikezwe ngemva kokwengeza i-lysate ukuze i-lyse ngokugcwele isampula.
(4) Uma usebenzisa indlela ye-Trizol ukuze ukhiphe, umgomo wokumunca amandla angaphezu kwavamile ngemva kokuhlukaniswa ngokuthi "ukhetha ukuhogela kancane kunokuhogela kakhulu", futhi akumele kukhishwe ungqimba olumaphakathi, ngaphandle kwalokho kuzodala ukungcoliswa okukhulu kwe-genomic DNA.
(5) Lapho ugeza, uketshezi lokuwasha kufanele lungene ngokugcwele eduze kodonga lweshubhu ukuze kuqinisekiswe ukuwashwa ngokuphelele.
(6) Ngendlela yokukhipha ikholomu, ngaphezu kokukhipha ikholomu ngemva kokugeza, ikholomu ye-adsorption kufanele futhi ibekwe ebhentshini elihlanzeke kakhulu futhi ifuthwe imizuzu engu-5-10 ukuze ihwamuke ngokugcwele isincibilikis esiphilayo sibe somile.
(7) Ekuhlaziyweni kokugcina kwendlela yekholomu, ngemva kokwengeza amanzi e-DEPC, kufanele ifukanyelwe imizuzu engu-3-5, noma amanzi e-DEPC kufanele ashise abe ngu-60 ° C kusengaphambili ukuze kwandiswe isivuno se-lution.Ngendlela yendabuko ye-Trizol cleavage kanye ne-isopropanol precipitation, i-RNA yokugcina incibilika emanzini e-DEPC, ngakho-ke kufanele kunikezwe isikhathi esifanele sokuhlakazwa, futhi phansi kweshubhu le-centrifuge kufanele kuqhunyiswe ngokuqhubekayo ithiphu le-pipette.
3 Three Izimbangela nezisombululo zokuhlushwa kwe-RNA ephansi/ikhwalithi embi
1. Isivuno siphansi kakhulu
Isampula ekhishiwe iphansi kakhulu, inani eliphelele alanele, noma isampula ekhishiwe sikhulu kakhulu futhi i-lysis ayiphelele;izicubu noma amangqamuzana ekhwalithi efanele kufanele asetshenziselwe ukukhishwa, ukwelashwa kwangaphambili kwesampula kufanele kwenziwe kahle, futhi i-lysis kufanele yanele.
2. Izinsalela ze-genome
Lapho ikhishwa ngendlela ye-Trizol, lapho i-supernatant imuncwa ungqimba oluphakathi nendawo ngemva kokwendlaleka, ukungcoliswa kofuzo okubi kakhulu kuzobangelwa;ukunakekelwa okwengeziwe kufanele kuthathwe uma layering ukugwema ukuncela phakathi ungqimba.Uma indlela yekholomu isetshenziselwa ukukhipha, ikhithi equkethe i-DNase I ingakhethwa ukuze ikhishwe.I-nucleic acid ekhangiswa kulwelwesi igaywa ngokuqondile nge-DNase I, enganciphisa kakhulu izinsalela ze-DNA.
3. Ukucekelwa phansi kwe-RNA
Kungase kube ukucekelwa phansi kwesampula ekhishiwe ngokwayo, noma ukucekelwa phansi okudalwe phakathi nenqubo yokukhipha;ngangokunokwenzeka, amasampula amasha kufanele asetshenziselwe ukukhipha i-RNA, futhi amasampula aqoqiwe kufanele agcinwe ku-nitrogen ewuketshezi noma -80°C esiqandisini ngesikhathi, futhi ukuqhwaza okuphindaphindiwe nokuncibilika kufanele kugwenywe.Amathiphu wamahhala we-RNase/DNase, amashubhu e-centrifuge nezinye izinto kufanele zisetshenziswe enqubweni yokukhipha i-RNA.Inqubo yokukhipha kufanele isheshe ngangokunokwenzeka.I-RNA ekhishiwe kufanele ibekwe ebhokisini leqhwa futhi igcinwe ku -80 ngesikhathi.Uma i-RNA ekhishiwe idinga ukutholwa ngejeli electrophoresis, i-electrophoresis kufanele yenziwe ngokushesha ngemva kokukhipha, futhi isigcinalwazi se-electrophoresis kufanele sishintshwe kufakwe esisha esilungisiwe.
4. Izinsalela zikasawoti kanye ne-organic solvent
Ama-reagents okukhipha aqukethe usawoti we-phenol ne-guanidine, futhi isisombululo sokugeza sine-ethanol.Phakathi nenqubo yokukhipha, i-lysate ayizange ifakwe ngokuphelele futhi ilahlwe, futhi isisombululo sokugeza asizange somiswe ngokugcwele.Usawoti oyinsalela nezincibilikisi zemvelo kuyingozi ekulotshweni okuhlanekezelwe okulandelayo kanye ne-PCR.Amadigri ahlukene okuvimbela, ngakho-ke i-lysate yezicubu kufanele isuswe ngokugcwele ngesikhathi senqubo yokukhipha, futhi ukugeza kufanele kube ngokwanele ukuze izindonga ezizungezile ze-tube zigezwe.Ngaphezu kwalokho, i-tube iyathululwa futhi ishaywa yisinyathelo esidingekayo, esizoqhubeka nokunciphisa insalela yezinto eziphilayo.
Ukuze uthole ulwazi olwengeziwe mayelana nokukhipha i-RNA, sicela ulandele iwebhusayithi yethu:
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Isikhathi sokuthumela: Dec-01-2022
