1. Menya kwinjiza igisubizo cya RNA
Absorbance kuri 280, 320, 230, na 260 nm yerekana indangagaciro za acide nucleic, background (igisubizo turbidity), kwibanda kumunyu, nibintu kama nka proteine.Mubisanzwe reba gusa OD260 / OD280 (Ikigereranyo, R).Iyo 1.8 ~ 2.0, twibwira ko kwanduza poroteyine cyangwa ibindi bintu kama muri RNA bishobora kwihanganira, ariko twakagombye kumenya ko mugihe Tris ikoreshwa nka buffer kugirango imenye iyinjira, agaciro R gashobora kuba karenze 2 (muri rusange kagomba kuba <2.2).Iyo R <1.8, umwanda wa poroteyine cyangwa ibindi bintu kama mubisubizo biragaragara cyane, kandi iherezo rya RNA rishobora kugenwa ukurikije ibikenewe.Iyo R> 2.2, bivuze ko RNA yahinduwe hydrolyz muri acide nucleic imwe.
2.Uburyo bwa elegitoronike ya RNA
Mubisanzwe, denaturing gel ikoreshwa kuri RNA electrophoreis, ariko niba ari iyo kumenya gusa ubwiza bwa RNA, gel gel ikenewe ntabwo ikenewe, kandi geli isanzwe ya agarose irashobora gukoreshwa.Intego ya electrophoreis ni ukumenya ubusugire bwa bande 28S na 18S nigipimo cyazo, cyangwa ubusugire bwa mRNA.Mubisanzwe, niba imirongo ya 28S na 18S ari nziza, isobanutse, kandi ityaye (bivuga ku mpande z’imigozi irasobanutse), kandi urumuri rwa 28S rurenze inshuro ebyiri urwa 18S, tubona ko ubwiza bwa RNA ari bwiza.
Ibyavuzwe haruguru nuburyo bubiri dukunze gukoresha, ariko murubwo buryo bubiri ntabwo bushobora kutubwira neza niba hari RNase isigaye mubisubizo bya RNA.Niba hari umubare muto cyane wa RNase mubisubizo, biragoye kuri twe kubimenya hamwe nuburyo bwavuzwe haruguru, ariko ibyinshi mubisubizo byimisemburo byakurikiyeho bikorwa hejuru ya dogere 37 kandi mugihe kirekire.Muri ubu buryo, niba hari umubare muto cyane wa RNase mubisubizo bya RNA, noneho hazabaho ibidukikije nigihe gikwiye cyo kugira uruhare rwabo mubushakashatsi bwakurikiyeho, kandi byanze bikunze ubushakashatsi buzaba bukonje muriki gihe.Hano hepfo turamenyekanisha uburyo bushobora kwemeza niba hari RNase isigaye mubisubizo bya RNA.
3. Ikizamini cyo kubungabunga ubushyuhe
Ukurikije urugero rwibanze, shushanya bibiri 1000 ng RNA mubisubizo bya RNA hanyuma ubyongereze kuri 0.5 ml centrifuge, hanyuma ubyongereze hamwe na pH 7.0 Tris buffer kugeza mubunini bwa 10 ul, hanyuma ushireho capa yigituba.Shyira kimwe muri byo mu bwogero bwamazi buhoraho kuri 70 ° C hanyuma ugumane ubushyuhe kuri 1 h.Ikindi gice cyabitswe muri firigo -20 ° C kuri 1 h.Igihe nikigera, kura ingero ebyiri za electrophorei.Nyuma ya electrophoreis irangiye, gereranya imirongo ya electrophoreque ya byombi.Niba imirwi yombi yombi idahuye cyangwa idafite itandukaniro rinini (birumvikana ko imirwi yabo nayo yujuje ibisabwa muburyo bwa 2), bivuze ko nta mwanda wa RNase usigaye mubisubizo bya RNA, kandi ubwiza bwa RNA nibyiza cyane.Ibinyuranye, niba icyitegererezo cyashyizwe kuri 70 ° C cyerekana kwangirika kugaragara, byerekana ko hari umwanda wa RNase mubisubizo bya RNA.
2 Uburyo bwubushakashatsi nubuhanga bwo gukuramo RNA
Ibibazo dukunze guhura nabyo mugihe dukuramo RNA ni: (1) umusaruro wa RNA ni muke;(2) RNA ifite umwanda mwinshi;(3) RNA ifite umwanda ukomeye wangiza;(4) icyitegererezo cyo gutesha agaciro nibindi bibazo
1. Bikunze gukoreshwa reagent zose za RNA
Uburyo bwa guanidine isothiocyanate hamwe nuburyo bwa Trizol nuburyo bukoreshwa cyane mugukuramo RNA yose mubice byinyamanswa ningirabuzimafatizo.Birakwiriye cyane cyane kuburugero ruto hamwe nuduce bigoye cyane kuvoma, nko gukuramo RNA yose kuruhu rwurukwavu hamwe ninyama zihuza inyamaswa;hiyongereyeho, Trizol, nka rusange ya lysis reagent, irashobora kandi gukoreshwa mugukuramo ibimera, bagiteri, ibihumyo nizindi ngingo.Kubice byibimera birimo polysaccharide na polifenol, nka camellia oleifera, amababi yicyayi, kungufu, nibindi, uburyo bwa CTAB burashobora kandi gukoreshwa mugukuramo RNA yose.
Nuburyo busanzwe, uburyo bwinkingi ebyiri nabwo burazwi cyane kubera imikorere yubushyuhe busanzwe, nta mpamvu yo kongeramo RNase, numutekano-nta chloroform, fenolisi nizindi ngirabuzimafatizo zo gukuramo.(ibicuruzwa bisabwa )
2. Gukuramo RNA yose mubice byinyamanswa
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.Tissue yakuweho igomba gutemagurwa byuzuye, igashyira ingirangingo zashwanyagujwe mu muyoboro wa EP udafite RNase, ukongeramo lysate, ingirangingo zacagaguye zigomba kuba zihuye neza na lysate, hanyuma zigategura homogenisation.
.Niba uturemangingo turimo poroteyine nyinshi, ibinure, cyangwa fibrous fibrous tissue nk'umwijima, ongera neza cyangwa ugabanye umubare wimyanya yaciwe (utabishaka) Hitamo 10 ~ 20 mg).
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(6) RNA yabonye nyuma yo gukuramo bwa nyuma igomba gushyirwa kumasanduku ya barafu kugirango igabanye kwangirika kwa RNA.
3. Gukuramo ingirabuzimafatizo ya RNA
.Ntukongere lysate mu ngirabuzimafatizo zaguye nyuma yo guta burundu amazi.Ibi bizatera paketi ya histone yasohotse nyuma ya lysed selile kurwego rwo hanze kwizirika hanze yingirabuzimafatizo, bityo bikagabanya guhuza ingirabuzimafatizo imbere muri pellet hamwe na lysate., bivamo lysis selile ituzuye kandi igabanya umusaruro wa RNA.
.Ongeramo lysate kuri EP tube ya enzyme yo gukuramo.
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4. Gutera RNA gukuramo
Utugingo ngengabuzima dukungahaye kuri fenolike, cyangwa ikungahaye kuri polysaccharide, cyangwa irimo metabolite ya kabiri itazwi, cyangwa ifite ibikorwa byinshi bya RNase.Ibi bintu byahujwe cyane na RNA nyuma ya lysis ya selile kugirango ibe ingirabuzimafatizo zidashonga cyangwa imvura igwa, bigoye kuyikuramo.Kubwibyo, iyo dukuyemo ibimera, dukeneye guhitamo ibikoresho byibimera.Lysate iri mu gikoresho irashobora gukemura neza ibibazo bya okiside yoroshye ya polifenol no gutandukanya ibice bya polysaccharide na acide nucleic.
(Kuri polysaccharide polifenol igihingwa cya RNA, ibicuruzwa bisabwa:
(1) Igishishwa, ifu, imbuto, amababi, nibindi byigihingwa bigomba kuba byuzuye mubutaka.Mugihe cyo gusya, azote yuzuye igomba kuzuzwa mugihe kugirango wirinde gushonga icyitegererezo.Icyitegererezo cyubutaka kigomba kongerwaho vuba muri lysate no kunyeganyezwa kugirango wirinde kwangirika kwa RNA.
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.Ibisanzwe bisanzwe byitwa homogenizers ntibishobora kuba ingirakamaro muguhuza ibimera, kandi mubisanzwe ntibisabwa.
5. Kwirinda gukuramo RNA
(1) Ingero z'imyenda zigomba kuba shyashya zishoboka kugirango wirinde gukonja no gukonja.
(2) Tissue igomba kuba yuzuye mugihe cyo kuyikuramo, kandi ingano yimyenda ntigomba kuba mike cyane, kereka cyane.
(3) Igihe gihagije cya incubation kigomba gutangwa nyuma yo kongeramo lysate kugirango lyse icyitegererezo.
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.Muburyo bwa Trizol clavage hamwe nuburyo bwo kugwa kwa isopropanol, RNA yanyuma isesekara mumazi ya DEPC, bityo rero hagomba gutangwa igihe gikwiye cyo guseswa, kandi hepfo yumuyoboro wa centrifuge ugomba guhora uhuhwa numuyoboro wa pipeti.
3 T.hree Impamvu nigisubizo cyo kwibanda kwa RNA nkeya / ubuziranenge
1. Umusaruro ni muke cyane
Icyitegererezo cyakuweho ni gito cyane, igiteranyo cyose ntigihagije, cyangwa icyitegererezo cyakuweho ni kinini kandi lysis ntabwo yuzuye;tissue cyangwa selile zifite ubuziranenge bukwiye gukoreshwa mugukuramo, mbere yo kuvura icyitegererezo bigomba gukorwa neza, kandi lysis igomba kuba ihagije.
2. Ibisigisigi bya genome
Mugihe cyo gukuramo nuburyo bwa Trizol, mugihe ndengakamere yinjijwe mugice cyo hagati nyuma yo guterana, hazaterwa kwandura genome zikomeye;hakwiye kwitabwaho cyane mugihe cyo kwirinda kugirango unywe mugice cyo hagati.Niba uburyo bwinkingi bwakoreshejwe mugukuramo, ibikoresho birimo DNase I nshobora gutoranywa kubikuramo.Acide nucleic yamamajwe kuri membrane igogorwa neza na DNase I, ishobora kugabanya cyane ibisigisigi bya ADN.
3. Kwangirika kwa RNA
Birashobora kuba ugutesha agaciro icyitegererezo cyakuweho ubwacyo, cyangwa gutesha agaciro kwatewe mugihe cyo gukuramo;uko bishoboka kwose, ingero nshya zigomba gukoreshwa mugukuramo RNA, kandi ingero zegeranijwe zigomba kubikwa muri firigo ya azote cyangwa -80 ° C mugihe gikwiye, kandi hagomba kwirindwa gukonjesha no gukonjesha inshuro nyinshi.RNase / DNase inama kubuntu, centrifuge tubi nibindi bikoresho bigomba gukoreshwa mugikorwa cyo gukuramo RNA.Igikorwa cyo gukuramo kigomba kwihuta bishoboka.RNA yakuweho igomba gushyirwa kumasanduku ya barafu ikabikwa kuri -80 mugihe.Niba RNA yakuweho igomba gutahurwa na gel electrophorei, electrophoreis igomba guhita ikorwa nyuma yo kuyikuramo, kandi buffer ya electrophoreis igomba gusimbuzwa iyindi yateguwe.
4. Ibisigazwa byumunyu nibinyabuzima
Imyanda ikuramo irimo fenol hamwe nu munyu wa guanidine, kandi igisubizo cyo gukaraba kirimo Ethanol.Mugihe cyo kuyikuramo, lysate ntiyigeze yinjizwa rwose kandi yarajugunywe, kandi igisubizo cyo gukaraba nticyumye neza.Umunyu usigaye hamwe nudukoko twangiza byangiza nyuma yo kwandukura hanyuma PCR.Inzego zitandukanye zo kubuza, bityo lysate ya tissue igomba kuvaho byuzuye mugihe cyo kuyikuramo, kandi gukaraba bigomba kuba bihagije kugirango inkuta zikikije umuyoboro zishobore gukaraba.Byongeye kandi, umuyoboro urimo ubusa kandi uhuhwa ni intambwe ikenewe, izarushaho kugabanya ibisigazwa by’ibinyabuzima.
Kubindi bisobanuro bijyanye no gukuramo RNA, nyamuneka kurikira urubuga rwacu:
www.foreivd.com kubindi bisobanuro.
Igihe cyo kohereza: Ukuboza-01-2022
