Ukwenziwa inzalo kwamathiphu e-pipette namashubhu e-EP, njll.
1. Lungiselela i-0.1% (inkulungwane eyodwa) i-DEPC (into enobuthi obuphezulu) ngamanzi angcolile, isebenzise ngokucophelela ku-fume hood, futhi uyigcine ku-4°C kude nokukhanya;
Amanzi e-DEPC amanzi ahlanzekile aphathwa nge-DEPC futhi afakwe inzalo ngokushisa okuphezulu nokucindezela okuphezulu.Ihlolelwe ukuthi ayinayo i-RNase, i-DNase ne-proteinase.
2. Faka ithiphu le-pipette neshubhu le-EP ku-0.1% DEPC, futhi uqinisekise ukuthi ithiphu ye-pipette neshubhu ye-EP kugcwaliswe ngo-0.1% DEP.
3. Vikela ekukhanyeni, ake ume, ubusuku bonke (12-24h)
4. Ibhokisi eliqukethe ithiphu neshubhu le-EP alidingi ukucwiliswa ku-DEPC.Ngemuva kokukhipha cishe amanzi e-DEPC ethiphuni noma kwishubhu le-EP, lipakishe bese uligoqa.
5. 121 degrees Celsius, 30min
6. 180 degrees Celsius, yome amahora ambalwa (okungenani amahora angu-3)
Qaphela: a.Gqoka amagilavu e-latex namamaski lapho uphatha i-DEPC!b, noma ngaphandle kokwenziwa inzalo kwe-DEPC, 130 ℃, 90min autoclave (ama-laboratories amaningi avala inzalo yokushisa okuphezulu kabili)
Izimpawu zokukhishwa kwe-RNA
Izimo ezimbili ezinkulu zokuhluleka kokuhlukaniswa kwezicubu kwe-RNA
Ukuwohloka kwe-RNA kanye nezinsalela zokungcola ezicutshini,mayelana nokucekelwa phansi, ake siqale sibheke ukuthi kungani i-RNA ekhishwe kumaseli akhulisiwe ingonakaliswa kalula.Ama-reagents akhipha i-RNA akhona wonke aqukethe izingxenye ezivimbela ngokushesha i-RNase.Faka i-lysate kumaseli akhulisiwe, futhi umane uxube, wonke amaseli angaxutshwa kahle ne-lysate, futhi amaseli ahlanjululwe ngokuphelele.Ngemuva kokuthi amaseli e-lysed, izithako ezisebenzayo ku-lysate zivimbela ngokushesha i-RNase ye-intracellular, ngakho i-RNA ihlala injalo.Okusho ukuthi, ngenxa yokuthi amangqamuzana akhulisiwe axhumene kalula futhi ngokugcwele ne-lysate, i-RNA yawo awonakali kalula;ngakolunye uhlangothi, i-RNA esicutshini yonakaliswa kalula ngoba amangqamuzana ezicubu akulula ukuxhumana ngokushesha ne-lysate.ngenxa yokuxhumana okwanele.Ngakho,sicabanga ukuthi kunendlela yokuguqula izicubu zibe ingqamuzana elilodwa kuyilapho kuvimbela umsebenzi we-RNA, inkinga yokonakala ingaxazululwa ngokuphelele.
Ukugaya i-nitrogen ewuketshezi kuyindlela enjalo ephumelela kakhulu.Kodwa-ke, indlela yokugaya i-nitrogen ewuketshezi inzima kakhulu, ikakhulukazi uma inani lamasampula likhulu.Lokhu kwaveza into engcono kakhulu elandelayo: i-homogenizer.Ii-homogenizerindlela ayicabangi umbuzo wokuthi umsebenzi we-RNase uvinjwa kanjani ngaphambi kokuthi amaseli axhunywe ne-lysate, kodwa kunalokho ithandazela ukuthi izinga lokuphazamiseka kwezicubu lisheshe kunezinga lapho i-intracellular RNase yehlisa isithunzi i-RN.
Umphumela we-homogenizer kagesi ungcono,futhi umphumela we-homogenizer yengilazi awulungile, kodwa ngokuvamile, indlela ye-homogenizer ayikwazi ukuvimbela into yokuwohloka.Ngakho-ke, uma isizinda sehlisiwe, i-homogenizer yokuqala kagesi kufanele isetshenziselwe ukugaya nge-nitrogen ewuketshezi;i-homogenizer yengilazi yokuqala kufanele ishintshwe ibe i-homogenizer kagesi noma igaywe ngokuqondile nge-nitrogen ewuketshezi.Inkinga cishe ingenzeka ngo-100%.zixazulule.
Inkinga yensalela yokungcola ethinta ukuhlolwa okulandelayo inezimbangela ezihluke kakhulu kunokonakaliswa, futhi izixazululo zihlukile ngokuhambisanayo.Ekuphetheni,uma kukhona ukuwohloka noma ukungcola okusele esicutshini, indlela yokukhipha/i-reagent yento ethile yokuhlola kufanele ithuthukiswe.Akudingeki ukuthi usebenzise amasampula akho ayigugu ukuze uthuthukise: ungathenga izilwane ezincane njengezinhlanzi/inkukhu emakethe, uthathe ingxenye ehambisanayo ye-RNA, kanye nengxenye yokukhipha amaprotheni - gaya ngomlomo, isisu kanye namathumbu Extract.
I-RNA eqondiwe ye-RNA ekhishiwe isetshenziselwa ukuhlola okuhlukile kokulandelela, futhi izidingo zayo zekhwalithi zihlukile
Ukwakhiwa komtapo wezincwadi we-cDNA kudinga ubuqotho be-RNA ngaphandle kwezinsalela ze-enzyme reaction inhibitors;INyakatho idinga ubuqotho be-RNA ephakeme kanye nezidingo eziphansi zezinsalela ze-enzyme reaction inhibitors;I-RT-PCR ayidingi ubuqotho be-RNA ephezulu kakhulu,kodwa ivimbela ukusabela kwe-enzyme.Izidingo zensalela ziqinile.Okokufaka kunquma okukhiphayo;ngaso sonke isikhathi umgomo uwukuthola ukuhlanzeka okuphezulu kwe-RNA, kuzobiza abantu nemali.
Ukuqoqwa/Ukugcinwa Kwamasampuli
Izinto Ezithinta Ukuwohloka Ngemva kokuba isampula lishiye umzimba ophilayo/noma indawo yokukhula yasekuqaleni, ama-enzyme angapheli kusampula azoqala ukonakalisa i-RNA,futhi izinga lokuwohloka lihlobene nokuqukethwe kwama-enzyme angapheli kanye nezinga lokushisa.Ngokwesiko, kunezindlela ezimbili kuphela zokuvimbela ngokuphelele umsebenzi we-endogenous enzyme: engeza i-lysate ngokushesha futhi i-homogenize kahle futhi ngokushesha;uthathe izingcezu ezincane futhi ngokushesha iqhwa in nitrogen liquid.Zombili izindlela zidinga ukusebenza ngokushesha.Eyakamuva ifanele wonke amasampula, kuyilapho eyokuqala ifaneleka kuphela izicubu ezinokuqukethwe okuphansi kwamaseli nama-endogenous enzyme futhi kulula ukwenza i-homogenize.Ngokukhethekile, izicubu zezitshalo, isibindi, i-thymus, amanyikwe, ubende, ubuchopho, amafutha, izicubu zemisipha, njll. ziqandiswe kangcono nge-nitrogen ewuketshezi ngaphambi kokuqhubeka.
Ukuhlukaniswa kanye ne-homogenization yamasampuli
Izinto Ezithinta Ukucekelwa phansi kanye Nokwehlukaniswa Kwesampula Kwesivunoukuze uthole i-homogenization ephelele, okokukhululwa okuphelele nokuphelele kwe-RNA.Amaseli angenziwa ngokuqondile ngaphandle kokuphulwa.Ama-tissue angahlukaniswa kuphela ngemuva kokuphulwa.Imvubelo namabhaktheriya adinga ukuphulwa ngama-enzyme ahambisanayo ngaphambi kokuthi ahlanganiswe.Izicubu ezinokuqukethwe kwe-endogenous endogenous ephansi kanye ne-homogenization elula ingachotshozwa futhi yenziwe i-homogenized ngesikhathi esisodwa ku-lysate nge-homogenizer;izicubu zezitshalo, isibindi, i-thymus, amanyikwe, ubende, ubuchopho, amafutha, izicubu zemisipha namanye amasampula, Aphezulu kuma-enzyme e-endo native noma awenziwa kalula njenge-homogenized,ngakho-ke ukuphazamiseka kwezicubu kanye ne-homogenization kumele kwenziwe ngokuhlukana.Indlela ethembeke kakhulu futhi ekhiqiza kakhulu yokuhlukanisa ukugaya nge-nitrogen ewuketshezi, futhi indlela enokwethenjelwa kakhulu ye-homogenization ukusetshenziswa kwe-homogenizer kagesi.Inothi elikhethekile mayelana nokugaya nge-nitrogen ewuketshezi: isampula akumele incibilike phakathi nayo yonke inqubo yokugaya, njengoba ama-enzyme e-endogenous cishe asebenze uma eqandisiwe.
Ukukhetha i-lysate
Okuthinta ukusebenza kalula kanye nezici zokungcola okusele kwe-endogenous Izixazululo ze-lysis ezivame ukusetshenziswa zingacishe zivimbele umsebenzi we-RNase.Ngakho-ke, iphuzu eliyinhloko lokukhetha isisombululo se-lysis ukucabangela ngokuhambisana nendlela yokuhlanza.Kukhona okuhlukile:amasampula anokuqukethwe okuphezulu kwe-endogenous enzyme anconywa ukuthi asebenzise i-lysate equkethe i-phenol ukuze kukhuliswe amandla okwenza ama-endogenous enzymes angasebenzi.
Ukukhetha indlela yokuhlanza
Izinto ezithinta ukungcola okusele kwe-endo native, isivinini sokukhipha Kumasampuli ahlanzekile njengamaseli, imiphumela egculisayo ingatholwa cishe nganoma iyiphi indlela yokuhlanza eseduze.Kodwa kwamanye amasampula amaningi, ikakhulukazi lawo anamazinga aphezulu okungcola njengezitshalo, isibindi, amagciwane, njll., ukukhetha indlela efanelekile yokuhlanza kubalulekile.Indlela yokuhlanza ikholomu ye-centrifugal inesivinini esisheshayo sokukhipha futhi ingasusa ngempumelelo ukungcola okuthinta ukusabela kwe-enzymatic okulandelayo kwe-RNA, kodwa kuyabiza(i-Forgene inganikeza amakhithi angabizi, chofoza eminye imininingwane.lapha);usebenzisa izindlela zokuhlanza ezongayo nezakudala, ezifana nemvula ye-LiCl, nakho kungathola imiphumela egculisayo, kodwa isikhathi sokusebenza side..
"Izigwegwe Ezintathu Nokunakwa Okuyisishiyagalombili" Ye-RNA Extraction
Isiyalo 1:Qeda ukungcoliswa kwama-exogenous enzymes.
Qaphela 1:Gqoka ngokuqinile imaski namagilavu.
Inothi 2:Amashubhu e-centrifuge, amakhanda amathiphu, izinduku ze-pipette, amathangi e-electrophoresis, namabhentshi okuhlola ahilelekile ekuhloleni kufanele alahlwe ngokuphelele.
Qaphela 3:Ama-reagents/izixazululo ezihilelekile ekuhloleni, ikakhulukazi amanzi, kumele angabi ne-RNAse.
Isiyalo 2:Vimba umsebenzi wama-endogenous enzymes
Inothi 4:Khetha indlela efanele ye-homogenization.
Inothi 5:Khetha i-lysate efanelekile.
Inothi 6:Lawula inani lokuqala lesampula.
Isiyalo 3:Cacisa inhloso yakho yokukhipha
Inothi 7:Nganoma iyiphi isistimu ye-lysate esondela enanini eliphezulu lokuqala lesampula, izinga lempumelelo yokukhipha lehla kakhulu.
Qaphela 8:Okuwukuphela kwendlela yezomnotho yokukhipha i-RNA ngempumelelo impumelelo ekuhloleni okwalandela, hhayi inzuzo.
Imithombo Ephezulu Eyishumi Yokungcola Kwe-RNAse
1. Iminwe ingumthombo wokuqala wama-enzyme angaphandle, ngakho-ke amagilavu kufanele agqokwe futhi ashintshwe njalo.Ngaphezu kwalokho, imaski kufanele igqokwe, ngoba ukuphefumula nakho kuwumthombo obalulekile wama-enzyme.Inzuzo eyengeziwe yokugqoka imaski yegilavu ukuvikela ohlolayo.
2. Amathiphu e-pipette, amashubhu e-centrifuge, ama-pipette - i-RNase ayikwazi ukwenziwa inzalo iyodwa, ngakho-ke amathiphu e-pipette namashubhu e-centrifuge kufanele aphathwe nge-DEPC, ngisho noma amakwe njenge-DEPC ephathwayo.Kungcono ukusebenzisa i-pipette enenhloso ekhethekile, uyisule ngebhola lekotini le-alcohol engu-75% ngaphambi kokusetshenziswa, ikakhulukazi induku;ngaphezu kwalokho, qiniseka ukuthi ungasebenzisi isikhiphi sekhanda.
3. Amanzi/isigcinalwazi kufanele singangcoliswa yi-RNase.
4. Okungenani itafula lokuhlola kufanele lisulwe ngamabhola kakotini otshwala angama-75%.
I-5.Endogenous RNase Zonke izicubu ziqukethe ama-enzyme e-endogenous, ngakho ukuqhwaza okusheshayo kwezicubu nge-nitrogen ewuketshezi kuyindlela engcono kakhulu yokunciphisa ukuwohloka.Indlela yokugcina i-nitrogen ewuketshezi/yokugaya ayilungile ngempela, kodwa iyona ndlela kuphela yezicubu ezinamazinga aphezulu ama-endogenous enzymes.
6. Amasampula e-RNA Imikhiqizo ekhipha i-RNA ingase iqukathe iminonjana yokungcola kwe-RNase.
7. Ukukhishwa kwe-Plasmid Ukukhishwa kwe-Plasmid kuvame ukusebenzisa i-Rnase ukuze kwehlise isithunzi se-RNA, futhi i-Rnase esele kufanele igaywe nge-Proteinase K futhi ikhishwe yi-PCI.
8. Ukugcinwa kwe-RNA Ngisho noma igcinwe ezingeni lokushisa eliphansi, ukulandelela amanani e-RNAse kuzodala ukuwohloka kwe-RNA.Isixazululo esingcono kakhulu sokulondolozwa kwesikhathi eside kwe-RNA ukumiswa kukasawoti/utshwala, ngoba utshwala buvimbela wonke umsebenzi we-enzymatic emazingeni okushisa aphansi.
9. Uma ama-cations (Ca, Mg) equkethe la ma-ion, ukushisa ku-80C imizuzu engu-5 kuzobangela ukuthi i-RNA iqhekeke, ngakho-ke uma i-RNA idinga ukushiswa, isixazululo sokulondoloza sidinga ukuqukatha i-chelating agent (1mM Sodium Citrate, pH 6.4).
10. Ama-enzyme asetshenziswa ekuhloleni okulandelayo angase angcoliswe i-RNase.
Amathiphu ayi-10 Okukhishwa kwe-RNA
1: Vimbela ngokushesha umsebenzi we-RNase.Amasampula aqandiswa ngokushesha ngemva kokuqoqwa, futhi i-RNase iyenziwa ingasebenzi ngokusebenza ngokushesha phakathi ne-lysis.
2: Khetha indlela efanelekile yokukhipha izicubu ezinokuqukethwe okuphezulu kwe-ribozyme, futhi izicubu ze-adipose kungcono ukusebenzisa indlela equkethe i-phenol.
3: Ikhwalithi yokubikezela idinga iNyakatho, ukwakhiwa komtapo wezincwadi we-cDNA kudinga ubuqotho obuphezulu, futhi i-RT-PCR ne-RPA (i-Ribonuclease protection assay) ayidingi ubuqotho obuphezulu.I-RT-PCR idinga ukuhlanzeka okuphezulu (izinsalela ze-enzyme inhibitor).
4: I-homogenization ephelele iyisihluthulelo sokuthuthukisa isivuno kanye nokunciphisa ukuwohloka.
5: Hlola ubuqotho bokutholwa kwe-RNA electrophoresis, 28S: 18S = 2: 1 uphawu oluphelele, 1: 1 futhi yamukelekile ekuhloleni okuningi.
I-6: Ukususwa kwe-DNA ye-RT-PCR, ukuhlaziywa kwe-array Kungcono ukusebenzisa i-Dnase I ukususa i-DNA.
I-7: Yehlisa ukungcoliswa kwama-enzyme angaphandle - ama-enzyme awakwazi ukungeniswa ngaphandle.
I-8: Uma ugxilisa i-nucleic acid ephansi, i-reagent co-precipitation kufanele yengezwe.Kodwa ukuvimbela i-co-precipitant equkethe ama-enzyme nokungcoliswa kwe-DNA.
9: Chaza kahle i-RNA, uma kunesidingo, shisa ku-65C imizuzu emi-5.
indlela yokugcina efanele
Ingagcinwa ku -20C isikhathi esifushane, futhi ku -80C isikhathi eside.Isinyathelo sokuqala sokuthuthukisa isivuno se-RNA ukubona ukuthi okuqukethwe kwe-RNA kwamasampuli ahlukene kuyehluka kakhulu.Ubuningi obukhulu (2-4ug/mg) njengesibindi, amanyikwe, inhliziyo, inala ephakathi (0.05-2ug/mg) njengobuchopho, umbungu, izinso, amaphaphu, i-thymus, i-ovary, ukuchichima okuphansi (<0.05ug/mg) mg) njengesinye, ithambo, amafutha.
1: Amaseli e-Lyse azokhipha i-RN - uma i-RNA ingakhululwa, isivuno sizoncishiswa.I-homogenization kagesi isebenza kangcono kunezinye izindlela ze-homogenization, kodwa ingase futhi idinge ukuhlanganiswa nezinye izindlela, njenge-liquid nitrogen mashing, ukugaya kwe-enzymatic (Lysozyme/Lyticase)
2: Ukuthuthukisa indlela yokukhipha.Izinkinga ezinkulu ngezindlela ezisekelwe ku-phenol ukuhlukaniswa okungaphelele kanye nokulahlekelwa kwe-RNA ingxenye (i-supernatant ayikwazi ukususwa ngokuphelele).Ukuhlukaniswa okungaphelele kungenxa ye-nucleic acid ephezulu kanye nokuqukethwe kwamaprotheni, okungaxazululwa ngokwandisa inani le-lysate elisetshenzisiwe noma ukunciphisa inani lesampula.Isinyathelo sokukhishwa kwe-chloroform sengezwe kuzicubu ze-adipose.Ukulahlekelwa kwe-RNA kungancishiswa ngokumpompa i-back-pump noma ngokususa ungqimba lwe-organic okulandelwa yi-centrifugation.Inkinga enkulu ngezindlela ezisuselwe kukholomu i-centrifugation isampula eyeqile.
Amathiphu Okukhipha Akudala
1. Ukuhlanzwa kwe-Phenol: Engeza umthamo olinganayo we-1: 1 Phenol / Chloroform bese uxuba ngamandla imizuzu engu-1-2.I-Centrifuge ngesivinini esikhulu imizuzu emi-2.Susa ngokucophelela i-supernatant (80-90%).Ungalokothi ufike esigabeni esiphakathi.Umthamo olinganayo wesixazululo sokusabela ungangezwa ku-Phenol/Chloroform futhi i-supernatant isuswe.Ama-supernatant amabili angaxutshwa ndawonye ukuze kube nemvula ye-nucleic acid ukuze kuthuthukiswe isivuno.Ungabi mnene kakhulu lapho uxuba, futhi ungazami ukususa wonke amandla angaphezu kwawemvelo.
2. Ukugeza nge-ethanol engu-70-80%: Ngesikhathi sokugeza, i-nucleic acid kufanele imiswe ukuze kuqinisekiswe ukuthi usawoti osele uyakhukhuleka.Ngesikhathi esifanayo, ngokushesha ngemva kokuthulula i-ethanol, i-centrifuge ngesivinini esikhulu imizuzwana embalwa, bese ususa i-ethanol esele nge-pipette.Chaza ngemva kokuma ekamelweni lokushisa imizuzu engu-5-10.
11. Ukukhishwa kwezinhlangano ezikhethekile
1. Izicubu ze-Fibrous: Isihluthulelo sokukhishwa kwe-RNA kusuka kuzicubu ze-fibrous ezifana nenhliziyo / imisipha yamathambo ukuphazamisa ngokuphelele izicubu.Lezi zicubu zine-cell density ephansi, ngakho-ke inani le-RNA ngeyunithi ngayinye yesisindo sezicubu liphansi, futhi kungcono kakhulu ukusebenzisa inani lokuqala eliningi ngangokunokwenzeka.Qinisekisa ukuthi ugaya izicubu kahle ngaphansi kwezimo eziqandisiwe.
2. Izicubu ezinamaphrotheni/amafutha aphezulu: okuqukethwe kwamafutha obuchopho/imifino kuphezulu.Ngemuva kokukhishwa kwe-PCI, i-supernatant iqukethe ama-floccules amhlophe.I-supernatant kufanele ikhishwe kabusha nge-chloroform.
3. Izicubu ezinokuqukethwe okuphezulu kwe-nucleic acid/ribozyme: i-spleen/thymus ine-nucleic acid ephezulu kanye nokuqukethwe kwe-ribozyme.Ukugaya izicubu ngaphansi kwezimo zokuqhwa okulandelwa i-homogenization esheshayo kungawenza angasebenzi ama-ribozyme.Kodwa-ke, uma i-lysate i-viscous kakhulu (ngenxa yokuqukethwe okuphezulu kwe-nucleic acid), ukukhishwa kwe-PCI ngeke kukwazi ukuhleleka ngokuphumelelayo;ukwengeza i-lysate eyengeziwe kungaxazulula le nkinga.Ukukhishwa kwe-PCI eminingi kungasusa i-DNA eyinsalela eyengeziwe.Uma imvula emhlophe iba ngokushesha ngemva kokwengeza utshwala, ibonisa ukungcoliswa kwe-DNA.Ukukhipha kabusha nge-PCI ene-acidic ngemva kokuhlakazwa kungasusa ukungcoliswa kwe-DNA.
4. Izicubu zezitshalo: Izicubu zezitshalo ziyinkimbinkimbi kunezicubu zezilwane.Ngokuvamile, izitshalo zigaywa ngaphansi kwezimo ze-nitrogen ewuketshezi, ngakho ukucekelwa phansi kwe-RNA ngama-endogenous enzymes akuvamile.Uma inkinga yokwehliswa kwesithunzi ingaxazululeki, cishe ibangelwa ukungcola okuqukethwe kusampula.Ukungcola okuqukethwe ezitshalweni eziningi kuzoholela ezinsaleleni, futhi isizathu sezinsalela ngokuvamile kungenxa yokuthi lokhu kungcola kunokufana okuthile ne-RNA: uyanetha bese mina ngiyanetha, futhi uyakhangisa futhi ngiyakukhangisa.Lezi zici zinquma ukuthi zingama-enzyme inhibitors aqine kakhulu.
Njengamanje, ama-reagents okukhishwa kwe-RNA okuhweba angashintshwa cishe kuzo zonke izicubu zezilwane ezinokulungiswa okuncane, kodwa kukhona ama-reagents okukhishwa kwe-RNA ambalwa angafanela izicubu eziningi zezitshalo.Ngenhlanhla, i-Foregene inganikeza okukhethekileizitshalo ze-RNA extraction kits, sineIkhithi yokuhlukanisa i-RNA Yezitshalo, Plant Total RNA Isolation kit Plus.Lesi sakamuva senzelwe izitshalo ezine-polysaccharide ephezulu kanye ne-polyphenol.Ngokukhishwa kwe-RNA, impendulo evela kubasebenzisi belebhu yinhle kakhulu.
12. Umthelela wesampula yokuqandisa nokuncibilika Isampuli eqandisiwe ingaba nkulu, futhi idinga ukusikwa ngaphambi kokuba isetshenziselwe ukukhipha i-RNA.Amasampula avame ukuncibilika (mhlawumbe ingxenye) ngesikhathi sokusika.Amasampula aqandisiwe angase adinge ukukalwa ngaphambi kokukhipha i-RNA, futhi ukuncibilika kuzokwenzeka ngempela phakathi nale nqubo.Ngezinye izikhathi, ukuncibilika kwesampula kwenzeka ngesikhathi senqubo yokugaya i-nitrogen;noma isampula eqandisiwe yengezwa ngokuqondile ku-lysate ngaphandle kokugaya i-nitrogen ewuketshezi, futhi ukuncibilika kuzokwenzeka ngaphambi kokuhlanganiswa okuphelele.Ukuhlola kubonise ukuthi izicubu eziqandisiwe zithambekele kakhulu ekonakaleni kwe-RNA ngesikhathi sokuncibilika kunezicubu ezintsha.Isizathu okungenzeka ukuthi: Inqubo yokuncibilikisa iqhwa iphazamisa izakhiwo ezingaphakathi kwengqamuzana, okwenza kube lula ngama-enzyme angaphakathi ukuthi athintane ngqo ne-RNA.
13. Ukwahlulela kwekhwalithi ye-RNA Ngokuvamile, i-electrophoresis isetshenziselwa ukwahlulela ubuqotho be-RNA, futhi i-A260/A280 isetshenziselwa ukwahlulela ubumsulwa be-RNA.Ngokombono, i-RNA engaguquki inesilinganiso esingu-28S:18S = 2.7:1, futhi idatha eminingi igcizelela isilinganiso sika-28S:18S = 2:1.Iqiniso liwukuthi cishe ayikho neyodwa ye-RNA ekhishwe kumasampuli ngaphandle kwamaseli esesilinganisweni esingu-2:1 (lokhu kutholwe kusetshenziswa i-Agilent Bioanalyzer).
Imiphumela ye-electrophoresis ye-RNA ithintwa izici eziningi, okuhlanganisa ukwakheka kwesibili, izimo ze-electrophoresis, umthwalo wesampula, izinga lokugcwala ngo-EB, njll. Sebenzisa i-electrophoresis yomdabu ukuze uthole i-RNA futhi usebenzise i-DNA Marker njengesilawuli.Uma i-28S kokuthi 2kb kanye ne-18S kokuthi 0.9kb icacile, futhi 28S: 18S > 1, ubuqotho bungahlangabezana nezimfuneko zokuhlolwa okuningi okwalandela.
I-A260/A280 iyinkomba edale ukudideka okukhulu.Okokuqala, kubalulekile ukucacisa incazelo yokuqala yale nkomba ye-nucleic acid: i-RNA ehlanzekile, i-A260/280 yayo = mayelana ne-2.0.I-RNA ehlanzekile 'yimbangela' futhi i-A260/A280 = 2 'umphumela'.Manje wonke umuntu usebenzisa i-A260/A280 'njengesizathu', ecabanga ukuthi “uma i-A260/A280 = 2, i-RNA imsulwa”, okuholela ngokwemvelo ekudidekeni.
Uma unentshisekelo, ungangeza i-reagent encane evame ukusetshenziswa ekukhishweni, njenge-phenol, i-guanidine isothiocyanate, i-PEG, njll., kusampula yakho ye-RNA, bese ukala isilinganiso se-A260/A280.Iqiniso liwukuthi ama-reagents amaningi asetshenziselwa ukukhipha i-RNA, kanye nokungcola okuningi kusampula, amunca cishe i-A260 ne-A280, ethinta i-A260/A280.
Indlela efundisa kakhulu njengamanje ukuskena amasampula e-RNA ebangeni elingu-200-300 nm.Ijika le-RNA emsulwa linezici ezilandelayo: ijika libushelelezi, i-A230 ne-A260 amaphuzu amabili okushintshashintsha, i-A300 iseduze no-0, A260/A280 = eduze kuka-2.0, kanye ne-A260/A230 = cishe ku-2.0.Uma idatha yokuskena ingatholakali, isilinganiso se-A260/A230 kufanele sinqunywe, njengoba lesi silinganiso sizwela kakhulu ekudluliseni kwakho konke ukungcola okuthinta ukusabela kwe-enzyme.Cabangela ububanzi bomugqa wedivayisi (0.1–0.5 ye-A260).
Kunezinye izenzakalo ezimbili eziwusizo: isilinganiso sizoba cishe ngo-0.3 ngaphansi uma i-A260/A280 ikalwa emanzini;kuyilapho isilinganiso esikalwa ngo-10 mM EDTA sicishe sibe ngu-0.2 ngaphezu kwalokho esikalwa ngo-1 mM EDTA.
Imikhiqizo ehlobene:
I-China Plant Total RNA Isolation Kit Umkhiqizi Nomhlinzeki |I-Foregene (foreivd.com)
Uchungechunge lokuhlukaniswa kwe-RNA - Foregene Co., Ltd. (foreivd.com)
Isikhathi sokuthumela: Jul-15-2022
