1. Dziwani kuyamwa kwa njira ya RNA
Absorbance pa 280, 320, 230, ndi 260 nm imayimira zofunikira za nucleic acid, maziko (solution turbidity), mchere wa mchere, ndi zinthu zamoyo monga mapuloteni, motero.Nthawi zambiri yang'anani pa OD260/OD280 (Ratio, R).Pamene 1.8 ~ 2.0, timaganiza kuti kuipitsidwa kwa mapuloteni kapena zinthu zina zakuthupi mu RNA zikhoza kulekerera, koma ziyenera kuzindikiridwa kuti pamene Tris imagwiritsidwa ntchito ngati buffer kuti azindikire kuyamwa, mtengo wa R ukhoza kukhala waukulu kuposa 2 (nthawi zambiri uyenera kukhala <2.2).Pamene R<1.8, kuipitsidwa kwa mapuloteni kapena zinthu zina zamoyo mu yankho zimakhala zoonekeratu, ndipo tsogolo la RNA likhoza kutsimikiziridwa malinga ndi zosowa.Pamene R> 2.2, zikutanthauza kuti RNA wakhala hydrolyzed mu nucleic acid imodzi.
2.Electrophoretic chitsanzo cha RNA
Nthawi zambiri, denaturing gel imagwiritsidwa ntchito pa RNA electrophoresis, koma ngati imangozindikira mtundu wa RNA, gel osakaniza sikofunikira, ndipo gel osakaniza agarose angagwiritsidwe ntchito.Cholinga cha electrophoresis ndikuzindikira kukhulupirika kwa magulu a 28S ndi 18S ndi chiŵerengero chawo, kapena kukhulupirika kwa mRNA smear.Nthawi zambiri, ngati magulu a 28S ndi 18S ndi owala, omveka bwino, komanso akuthwa (ponena za m'mphepete mwa maguluwo ndi omveka bwino), ndipo kuwala kwa 28S kumaposa kuwirikiza kawiri kwa gulu la 18S, timawona kuti khalidwe la RNA ndi labwino.
Zomwe zili pamwambazi ndi njira ziwiri zomwe timakonda kugwiritsa ntchito, koma palibe njira ziwirizi zomwe zingatiuze momveka bwino ngati pali RNase yotsalira mu njira ya RNA.Ngati pali kachulukidwe kakang'ono ka RNase mu yankho, zimakhala zovuta kuti tizindikire ndi njira yomwe ili pamwambayi, koma zambiri zomwe zimatsatira za enzymatic zimachitika pamwamba pa madigiri 37 komanso kwa nthawi yaitali.Mwa njira iyi, ngati pali chiwerengero chochepa kwambiri cha RNase mu njira ya RNA, ndiye kuti padzakhala malo abwino kwambiri ndi nthawi yochita nawo gawo lawo pazoyesera zotsatila, ndipo ndithudi kuyesa kudzakhala kozizira panthawiyi.Pansipa tikuwonetsa njira yomwe ingatsimikizire ngati pali RNase yotsalira mu njira ya RNA.
3. Mayeso oteteza kutentha
Malinga ndi ndende ya zitsanzo, jambulani awiri a 1000 ng RNA kuchokera ku njira ya RNA ndikuwonjezera ku chubu cha 0,5 ml centrifuge, ndikuwonjezera ndi pH 7.0 Tris buffer ku chiwerengero cha 10 ul, ndiyeno musindikize kapu ya chubu.Ikani imodzi mwa madzi osamba osamba kutentha kwa 70 ° C ndikutentha kwa 1 h.Gawo lina linasungidwa mufiriji -20 ° C kwa 1 h.Nthawi ikatha, chotsani zitsanzo ziwiri za electrophoresis.Pambuyo pa electrophoresis, yerekezerani magulu a electrophoretic awiriwo.Ngati magulu awiriwa akugwirizana kapena alibe kusiyana kwakukulu (zowonadi, magulu awo amakumananso ndi zomwe zili mu njira 2), zikutanthauza kuti palibe kuipitsidwa kwa RNase yotsalira mu njira ya RNA, ndipo ubwino wa RNA ndi wabwino kwambiri.M'malo mwake, ngati chitsanzocho chikuphatikizidwa pa 70 ° C chikuwonetsa kuwonongeka koonekeratu, zimasonyeza kuti pali kuipitsidwa kwa RNase mu njira ya RNA.
2 Njira zoyesera ndi njira zochotsera RNA
Mavuto omwe timakumana nawo nthawi zambiri pochotsa RNA ndi awa: (1) Zokolola za RNA ndizochepa;(2) RNA ili ndi vuto lalikulu la mchere;(3) RNA ili ndi kuipitsidwa kwakukulu kwa organic zosungunulira;(4) kuwonongeka kwa zitsanzo ndi mavuto ena
1. Ambiri ntchito okwana RNA m'zigawo reagents
Njira ya guanidine isothiocyanate ndi njira ya Trizol ndiyo njira zomwe zimagwiritsidwa ntchito kwambiri pochotsa RNA yonse kuchokera kumagulu a nyama ndi maselo a nyama.Ndizoyenera makamaka kwa zitsanzo zazing'ono ndi minofu yomwe imakhala yovuta kwambiri kuchotsa, monga kuchotsa RNA yonse kuchokera ku khungu la kalulu ndi minofu yolumikizira nyama;Komanso, Trizol, monga ambiri-cholinga lysis reagent, Angagwiritsidwenso ntchito kwa m'zigawo za zomera, mabakiteriya, bowa ndi zimakhala.Kwa minyewa yazomera yokhala ndi ma polysaccharides ndi ma polyphenols, monga camellia oleifera, masamba a tiyi, rapeseed, ndi zina zotero, njira ya CTAB ingagwiritsidwenso ntchito kuchotsa RNA yonse.
Monga njira yodziwika bwino, njira yamitundu iwiri imakhalanso yotchuka kwambiri chifukwa cha kutentha kwake kwanthawi zonse, palibe chifukwa chowonjezera RNase, ndi chitetezo-palibe chloroform, phenols ndi ma reagents ena achilengedwe kuti achotse.(analimbikitsa mankhwala )
2. Kutulutsa RNA yonse kuchokera kumagulu a nyama
(1) Yesani kusankha minofu yatsopano, ngati si yatsopano (makamaka mkati mwa miyezi itatu - 80 ℃ firiji kapena chisanu mumadzi a nayitrogeni. Mukadula minofu, musadule mwachindunji kutentha kwa chipinda, onetsetsani kuti Muyike pa ayezi, yesetsani kupewa kuzizira mobwerezabwereza ndi kusungunuka.
(2) Gwiritsani ntchito lumo ndi ma tweezers oyera kuti mudule kachidutswa kakang’ono, yesetsani kudula mbali yapakati ya minyewayo podula chitsanzo, kapena kaye mudule kachidutswa kakang’ono kamene kali pakati, ndiyeno mudule chitsanzocho pamalo atsopanowo.Minofu yochotsedwa iyenera kuphwanyidwa bwino, ikani minofu yowonongeka mu chubu cha EP popanda RNase, onjezerani lysate, minofu yowonongeka iyenera kuwonetseredwa bwino ndi lysate, ndikukonzekera homogenization.
(3) Kwa minyewa yodziwika bwino, sankhani minyewa ya mung (30-60 mg) ya homogenization.Ngati minyewayi ili ndi mapuloteni ambiri, mafuta, kapena minyewa yolimba ngati chiwindi, moyenerera onjezerani kapena kuchepetsa kuchuluka kwa minyewa yodulidwa (posankha) Sankhani 10 ~ 20 mg).
(4) Ngati minofu ya nsomba, nyama ya shrimp, jellyfish ndi minyewa ina yokhala ndi madzi ochulukirapo imachotsedwa, kuchuluka kwachitsanzo kuyenera kukulitsidwa moyenera (kukulimbikitsidwa 100-200 mg).
(5) Ngati zinthu ziloleza, minofu ya nyama imatha kuchotsedwa mwachindunji pambuyo pa homogenized ndi homogenizer yamtundu wapamwamba, ngati palibe zida zotere.
(6) RNA yomwe imapezeka pambuyo pochotsa komaliza iyenera kuikidwa pa ayezi nthawi yomweyo kuti ichepetse kuwonongeka kwa RNA.
3. Maselo a RNA a zinyama
(1) Maselo oyimitsidwa: centrifuge mwachindunji ndi kutaya sing'anga, kusamba ndi wosabala PBS kwa nthawi 1-2, ndiye kuyimitsa ndi mlingo woyenera wa PBS, ndiyeno kuwonjezera lysate kwa lysis.Osawonjezera lysate mwachindunji ku ma cell omwe amalowa mutatha kutaya madziwo.Izi zidzachititsa kuti histone phukusi anamasulidwa pambuyo maselo lysed pa wosanjikiza akunja kumamatira kunja kwa maselo precipitated, potero kuchepetsa kukhudzana kwa maselo mkati pellet ndi lysate., zomwe zimapangitsa kuti maselo asamalire bwino komanso kuchepetsa zokolola za RNA.
(2) Maselo omwe ali ndi theka-otsatira kapena osatsatira mwamphamvu: Mukataya sing'angayo, yambani ndi PBS kwa nthawi 1-2, kenaka mutengere mlingo woyenera wa PBS ndikuwomba mbale ya chikhalidwe ndi pipette kapena mfuti kuti muwombe maselo, ndikuwasamutsira ku maselo opanda RNA.Onjezani lysate ku chubu cha EP cha enzyme kuti muchotse.
(3) Maselo ogwirizana: amayenera kudyetsedwa ndi trypsin poyamba, kenako amasonkhanitsidwa mu machubu a EP opanda RNase, centrifuged kuchotsa supernatant, kutsukidwa 1-2 nthawi ndi PBS kuchotsa trypsin owonjezera, ndi resuspended ndi mlingo woyenera wa PBS Kenako pitirizani sitepe yochotsa.
4. Bzalani RNA m'zigawo
Mitengo ya zomera imakhala ndi mankhwala ambiri a phenolic, kapena olemera mu polysaccharides, kapena imakhala ndi metabolites yachiwiri yosadziwika, kapena imakhala ndi zochita zambiri za RNase.Zinthuzi zimaphatikizidwa mwamphamvu ndi RNA pambuyo pa cell lysis kuti apange ma insoluble complexes kapena colloidal precipitates, omwe ndi ovuta kuchotsa.Choncho, tikamachotsa minofu ya zomera, tiyenera kusankha zida za zomera.The lysate mu zida akhoza bwino kuthetsa mavuto oxidation zosavuta wa polyphenols ndi kulekana kwa mankhwala polysaccharide ndi nucleic zidulo.
(Pa polysaccharide polyphenol chomera RNA m'zigawo, analimbikitsa mankhwala:
(1) Pepala, zamkati, njere, masamba, ndi zina zotero za mmera ziyenera kudulidwa mumtondo.Panthawi yopera, nayitrogeni wamadzimadzi uyenera kuwonjezeredwa panthawi yake kuti asasungunuke.Chitsanzo cha pansi chiyenera kuwonjezeredwa mwamsanga ku lysate ndikugwedezeka kuti tipewe kuwonongeka kwa RNA.
(2) Kwa zitsanzo za fiber monga mpunga ndi masamba a tirigu, kuchuluka kwa m'zigawo kuyenera kuchepetsedwa moyenerera, mwinamwake minofu ikupera ndi lysis sizidzatha, zomwe zimapangitsa kuti pakhale zokolola zochepa za RNA yotengedwa.
(3) Pamitengo yamitengo yokhala ndi madzi ambiri, monga zipatso za makangaza, zipatso za mavwende, zipatso za pichesi, ndi zina zotero, kukula kwachitsanzo kuyenera kukulitsidwa moyenera (100-200 mg ndiyosankha).
(4) Tizilombo tomera, monga masamba a mbewu, ma rhizomes, zipatso zolimba ndi zinthu zina zimalimbikitsidwa kugwiritsa ntchito nayitrogeni wamadzimadzi kuti asungunuke bwino zinthuzo mumtondo, ndikupita ku sitepe yochotsa.Ochiritsira minofu homogenizers sangakhale ogwira homogenizing zomera zimakhala, ndipo zambiri osavomerezeka.
5. Kusamala kwa RNA m'zigawo
(1) Zitsanzo za minofu ziyenera kukhala zatsopano momwe zingathere kuti zisawonongeke mobwerezabwereza ndi kusungunuka.
(2) Mnofuwo uyenera kuphwanyidwa bwino pouchotsa, ndipo kuchuluka kwa minofu sikuyenera kukhala kochepa kwambiri, ngakhale kuchulukirachulukira.
(3) Nthawi yokwanira yopangira makulitsidwe iyenera kuperekedwa mutatha kuwonjezera lysate kuti muyese bwino chitsanzo.
(4) Mukamagwiritsa ntchito njira ya Trizol kuti muchotse, mfundo yotengera mphamvu yamphamvu pambuyo pa stratification "ndimakonda kutulutsa mpweya pang'ono kuposa kutulutsa zambiri", ndipo sayenera kuchotsa pakati, apo ayi zidzayambitsa kuipitsidwa kwa DNA ya genomic.
(5) Potsuka, madzi ochapira ayenera kulowa mozungulira khoma la chubu kuti atsimikize kuti atsuka bwino.
(6) Pakuti mzati njira m'zigawo, kuwonjezera detaching ndime pambuyo kutsuka, ndime adsorption ayenera kuikidwa mu kopitilira muyeso-oyera benchi ndi kuwomberedwa kwa mphindi 5-10 kuti asamasanduke nthunzi zosungunulira organic kuti dryness.
(7) Pakutulutsa komaliza kwa njira yazanja, mutatha kuwonjezera madzi a DEPC, iyenera kuyikidwa kwa mphindi 3-5, kapena madzi a DEPC ayenera kutenthedwa mpaka 60 ° C pasadakhale kuti awonjezere zokolola za elution.Mu njira yachikhalidwe ya Trizol cleavage ndi isopropanol precipitation, RNA yomaliza imasungunuka m'madzi a DEPC, kotero nthawi yoyenera iyenera kuperekedwa kuti iwonongeke, ndipo pansi pa chubu cha centrifuge chiyenera kuwombedwa mosalekeza ndi nsonga ya pipette.
3 Three Zomwe Zimayambitsa ndi njira zothetsera ndende ya RNA / khalidwe losauka
1. Zokolola ndizochepa kwambiri
Chitsanzo chochotsedwacho ndi chochepa kwambiri, chiwerengero chonsecho sichikwanira, kapena chitsanzo chochotsedwa ndi chochuluka ndipo lysis siili yokwanira;minofu kapena maselo a khalidwe loyenerera ayenera kugwiritsidwa ntchito pochotsa, chithandizo chamankhwala chisanachitike chiyenera kuchitidwa bwino, ndipo lysis iyenera kukhala yokwanira.
2. Zotsalira za ma genome
Pochotsa ndi njira ya Trizol, pamene chowonjezeracho chimayamwa pakati pa kusanjikiza, kuipitsidwa kwakukulu kwa matupi athu kudzayamba;chisamaliro chowonjezera ayenera kuchitidwa pamene wosanjikiza kupewa kuyamwa pakati wosanjikiza.Ngati njira ya mzati ikugwiritsidwa ntchito pochotsa, zida zomwe zili ndi DNase I zitha kusankhidwa kuti zichotsedwe.The nucleic acid adsorbed pa nembanemba imagayidwa mwachindunji ndi DNase I, yomwe ingachepetse kwambiri zotsalira za DNA.
3. Kuwonongeka kwa RNA
Kungakhale kuwonongeka kwa chitsanzo chochotsedwa chokha, kapena kuwonongeka komwe kumachitika panthawi yochotsa;momwe zingathere, zitsanzo zatsopano ziyenera kugwiritsidwa ntchito pochotsa RNA, ndipo zitsanzo zomwe zasonkhanitsidwa ziyenera kusungidwa mumadzi a nayitrogeni kapena -80 ° C mufiriji panthawi yake, ndipo kuzizira kobwerezabwereza ndi kusungunuka kuyenera kupewedwa.Malangizo aulere a RNase/DNase, machubu a centrifuge ndi zida zina ziyenera kugwiritsidwa ntchito pochotsa RNA.Njira yochotsera iyenera kukhala yofulumira kwambiri.RNA yotengedwa iyenera kuikidwa pa ayezi ndikusungidwa pa -80 nthawi.Ngati RNA yotengedwa iyenera kuzindikiridwa ndi gel electrophoresis, electrophoresis iyenera kuchitidwa mwamsanga pambuyo pochotsa, ndipo electrophoresis bafa iyenera kusinthidwa ndi yokonzedwa kumene.
4. Zotsalira za mchere ndi organic zosungunulira
The reagents m'zigawo muli phenol ndi guanidine mchere, ndi kutsuka njira lili Mowa.Panthawi yochotsa, lysate sinatengedwe kwathunthu ndikutayidwa, ndipo njira yotsuka sinaume kwathunthu.Mchere wotsalira ndi zosungunulira za organic ndizowopsa pakulemba kotsatira ndi PCR.Madigiri osiyanasiyana oletsa, kotero minofu ya lysate iyenera kuchotsedwa kwathunthu panthawi yochotsa, ndipo kutsuka kuyenera kukhala kokwanira kuti makoma ozungulira a chubu atsukidwe.Kuonjezera apo, chubucho chimachotsedwa ndikuwombedwa ndi sitepe yofunikira, yomwe idzachepetsenso zotsalira za organic.
Kuti mumve zambiri za kuchotsedwa kwa RNA, chonde tsatirani tsamba lathu:
www.foreivd.com kuti mumve zambiri.
Nthawi yotumiza: Dec-01-2022
