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1. Tshawb xyuas qhov nqus ntawm RNA kua

Absorbance ntawm 280, 320, 230, thiab 260 nm sawv cev rau cov txiaj ntsig ntawm nucleic acid, keeb kwm yav dhau (kev daws teeb meem), ntsev concentration, thiab cov organic teeb meem xws li protein, feem.Feem ntau tsuas saib OD260/OD280 (Ratio, R).Thaum 1.8 ~ 2.0, peb xav tias cov kab mob sib kis ntawm cov protein lossis lwm yam khoom siv hauv RNA tuaj yeem zam tau, tab sis nws yuav tsum tau muab sau tseg tias thaum Tris siv los ua qhov tsis pom qhov nqus, tus nqi R yuav ntau dua 2 (feem ntau nws yuav tsum yog <2.2).Thaum R <1.8, cov pa phem ntawm cov protein los yog lwm yam organic teeb meem hauv kev daws teeb meem yog pom tseeb dua, thiab txoj hmoo ntawm RNA tuaj yeem txiav txim siab raws li qhov xav tau.Thaum R> 2.2, nws txhais tau tias RNA tau hydrolyzed rau hauv ib qho nucleic acid.
 
2.Electrophoretic qauv ntawm RNA
Feem ntau, denaturing gel yog siv rau RNA electrophoresis, tab sis yog tias nws tsuas yog txhawm rau kuaj qhov zoo ntawm RNA, denaturing gel tsis tsim nyog, thiab cov gel zoo tib yam tuaj yeem siv tau.Lub hom phiaj ntawm electrophoresis yog txhawm rau txheeb xyuas qhov kev ncaj ncees ntawm 28S thiab 18S bands thiab lawv qhov sib piv, lossis kev ncaj ncees ntawm mRNA smear.Feem ntau, yog tias 28S thiab 18S bands ci ntsa iab, meej, thiab ntse (hais txog cov npoo ntawm cov bands meej), thiab qhov ci ntawm 28S yog ntau tshaj ob npaug ntawm 18S band, peb xav txog qhov zoo ntawm RNA kom zoo.
Cov saum toj no yog ob txoj hauv kev uas peb nquag siv, tab sis ob qho ntawm ob txoj kev no tsis tuaj yeem qhia peb seb puas muaj RNase seem hauv RNA daws.Yog hais tias muaj tsawg heev ntawm RNase nyob rau hauv cov tshuaj, nws yog ib qho nyuaj rau peb mus ntes nws nrog cov saum toj no txoj kev, tab sis feem ntau ntawm cov tom ntej enzymatic cov tshuaj tiv thaiv yog nqa tawm nyob rau saum toj 37 degrees thiab ntev.Nyob rau hauv txoj kev no, yog tias muaj tsawg heev ntawm RNase hauv cov tshuaj RNA, ces yuav muaj ib qho chaw tsim nyog thiab lub sijhawm los ua lawv lub luag haujlwm hauv cov kev sim tom ntej, thiab tau kawg qhov kev sim yuav txias rau lub sijhawm no.Hauv qab no peb qhia txog ib txoj hauv kev uas tuaj yeem paub meej tias puas muaj RNase seem hauv cov tshuaj RNA.
 
3. Kev ntsuas kub ceev
Raws li cov qauv concentration, kos ob 1000 ng RNA los ntawm RNA tov thiab ntxiv rau 0.5 ml centrifuge raj, thiab ntxiv nws nrog pH 7.0 Tris tsis rau tag nrho cov ntim ntawm 10 ul, thiab ces kaw lub hau ntawm lub raj.Muab ib qho ntawm lawv tso rau hauv ib da dej kub tas li ntawm 70 ° C thiab ua kom sov li 1 teev.Lwm qhov yog khaws cia rau hauv -20 ° C lub tub yees rau 1 h.Thaum lub sijhawm nce, tshem tawm ob qho qauv rau electrophoresis.Tom qab electrophoresis tiav lawm, sib piv cov electrophoretic bands ntawm ob.Yog tias cov bands ntawm ob qhov zoo ib yam lossis tsis muaj qhov sib txawv tseem ceeb (qhov tseeb, lawv cov bands kuj ua tau raws li cov xwm txheej hauv txoj kev 2), nws txhais tau hais tias tsis muaj RNase seem hauv cov tshuaj RNA, thiab qhov zoo ntawm RNA yog qhov zoo heev.Ntawm qhov tsis sib xws, yog tias tus qauv incubated ntawm 70 ° C qhia pom kev degradation, nws qhia tau hais tias muaj RNase kab mob hauv cov tshuaj RNA.
 
2 Kev sim thiab cov txheej txheem rau RNA rho tawm
Cov teeb meem peb feem ntau ntsib thaum rho tawm RNA yog: (1) RNA tawm los tsawg;(2) RNA muaj kuab paug hnyav heev;(3) RNA muaj cov kuab tshuaj organic hnyav;(4) qauv degradation thiab lwm yam teeb meem
 
1. Feem ntau siv tag nrho RNA extraction reagents
Txoj kev guanidine isothiocyanate thiab txoj kev Trizol yog txoj kev siv ntau tshaj plaws rau kev rho tawm tag nrho RNA los ntawm cov ntaub so ntswg tsiaj thiab cov tsiaj txhu.Nws yog tshwj xeeb tshaj yog haum rau cov qauv me me thiab cov ntaub so ntswg uas tshwj xeeb tshaj yog nyuaj rau rho tawm, xws li rho tawm tag nrho RNA los ntawm cov tawv nqaij luav thiab cov tsiaj sib txuas;Tsis tas li ntawd, Trizol, raws li lub hom phiaj dav dav lysis reagent, kuj tuaj yeem siv rau kev tshem tawm cov ntaub so ntswg, kab mob, fungi thiab lwm yam ntaub so ntswg.Rau cov ntaub so ntswg uas muaj polysaccharides thiab polyphenols, xws li camellia oleifera, tshuaj yej nplooj, rapeseed, thiab lwm yam., CTAB txoj kev kuj tseem siv tau los rho tawm tag nrho RNA.

Raws li ib qho piv txwv, txoj kev ob-kem kuj tseem nrov heev vim nws qhov kev ua haujlwm kub ib txwm, tsis tas yuav ntxiv RNase, thiab kev nyab xeeb - tsis muaj chloroform, phenols thiab lwm yam organic reagents rau extraction.(cov khoom pom zoo )

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2. Kev rho tawm tag nrho RNA los ntawm cov ntaub so ntswg tsiaj
 
(1) Sim xaiv cov ntaub so ntswg tshiab, yog tias nws tsis tshiab (zoo dua li ntawm peb lub hlis - 80 ℃ lub tub yees los yog khov hauv cov kua nitrogen. Thaum txiav cov ntaub so ntswg, tsis txhob txiav ncaj qha rau ntawm chav tsev kub, nco ntsoov muab tso rau hauv lub thawv dej khov, sim kom tsis txhob rov khov thiab thawing.
(2) Siv cov txiab huv si thiab tweezers txiav ib daim me me ntawm cov ntaub so ntswg, sim txiav qhov nruab nrab ntawm cov ntaub so ntswg thaum txiav cov qauv, los yog thawj zaug txiav cov ntaub so ntswg loj los ntawm nruab nrab, thiab tom qab ntawd txiav cov qauv ntawm qhov tshiab incision.Cov ntaub so ntswg tshem tawm yuav tsum tau muab tshem tawm tag nrho, muab cov ntaub so ntswg tso rau hauv EP raj yam tsis muaj RNase, ntxiv cov lysate, cov ntaub so ntswg shredded yuav tsum tau tag nrho rau cov lysate, thiab npaj rau homogenization.

(3) Rau cov ntaub so ntswg ib txwm, xaiv cov ntaub so ntswg mung taum loj (30-60 mg) rau homogenization.Yog tias cov ntaub so ntswg muaj cov protein ntau, rog, lossis cov ntaub so ntswg ntom ntom xws li daim siab, tsim nyog nce lossis txo cov ntaub so ntswg (yeem) Xaiv 10 ~ 20 mg).
(4) Yog tias cov nqaij ntses, cov nqaij cw, jellyfish thiab lwm cov ntaub so ntswg uas muaj dej ntau tau muab rho tawm, cov qauv ntim yuav tsum tau nce ntxiv (pom zoo 100-200 mg).
(5) Yog tias muaj kev tso cai, cov ntaub so ntswg tuaj yeem raug rho tawm ncaj qha tom qab ua homogenized nrog cov ntaub so ntswg siab homogenizer, yog tias tsis muaj cov khoom siv zoo li no.
(6) RNA tau txais tom qab kev rho tawm zaum kawg yuav tsum muab tso rau ntawm lub thawv dej khov tam sim ntawd kom txo tau qhov degradation ntawm RNA.

3. Tsiaj cell RNA rho tawm

(1) Tshem tawm cov hlwb: centrifuge ncaj qha thiab pov tseg qhov nruab nrab, ntxuav nrog tsis muaj menyuam PBS rau 1-2 zaug, tom qab ntawd tshem tawm nrog qhov tsim nyog ntawm PBS, thiab tom qab ntawd ntxiv lysate rau lysis.Tsis txhob ntxiv cov lysate ncaj qha mus rau lub hlwb precipitated tom qab tag nrho cov kua pov tseg.Qhov no yuav ua rau lub pob histone tso tawm tom qab cov lysed hlwb ntawm txheej txheej kom ua raws li sab nraud ntawm cov hlwb precipitated, yog li txwv kev sib cuag ntawm cov hlwb hauv cov pellet nrog cov lysate., ua rau tsis tiav cell lysis thiab txo RNA tawm los.

(2) Cells uas yog ib nrab los yog tsis nruj me ntsis adherent: Tom qab muab pov tseg qhov nruab nrab, ntxuav nrog PBS rau 1-2 lub sij hawm, ces ncaj qha nqus ib tug npaum li cas ntawm PBS thiab tshuab cov kab lis kev cai tais nrog ib tug pipette los yog rab phom los tshuab tawm lub hlwb, thiab hloov mus rau RNA-dawb hlwb.Ntxiv cov lysate rau EP raj ntawm cov enzyme rau kev rho tawm.

(3) Adherent cells: yuav tsum tau zom nrog trypsin ua ntej, tom qab ntawd sau rau hauv RNase-dawb EP raj, centrifuged tshem tawm cov supernatant, ntxuav 1-2 zaug nrog PBS kom tshem tawm cov trypsin ntau dhau, thiab rov ua dua nrog tus nqi tsim nyog ntawm PBS Tom qab ntawd mus rau theem extraction.

4. Cog RNA rho tawm

Cog cov ntaub so ntswg yog nplua nuj nyob rau hauv phenolic tebchaw, los yog nplua nuj nyob rau hauv polysaccharides, los yog muaj ib co unidentified thib ob metabolites, los yog muaj kev ua haujlwm siab ntawm RNase.Cov tshuaj no yog nruj nreem ua ke nrog RNA tom qab cell lysis los tsim insoluble complexes los yog colloidal precipitates, uas yog nyuaj rau tshem tawm.Yog li ntawd, thaum peb rho tawm cov ntaub so ntswg, peb yuav tsum xaiv cov khoom siv rau cov nroj tsuag.Cov lysate hauv cov khoom siv tuaj yeem daws cov teeb meem ntawm kev yooj yim oxidation ntawm polyphenols thiab kev sib cais ntawm polysaccharide tebchaw thiab nucleic acids.

(Rau polysaccharide polyphenol cog RNA extraction, pom zoo cov khoom:

(1) Lub tev, pulp, noob, nplooj, thiab lwm yam ntawm cov nroj tsuag yuav tsum tau muab tso rau hauv ib lub mortar.Thaum lub sij hawm sib tsoo, cov kua nitrogen yuav tsum tau muab ntxiv rau lub sijhawm kom tsis txhob yaj cov qauv.Cov qauv hauv av yuav tsum tau ntxiv sai sai rau lysate thiab shaken kom tsis txhob RNA degradation.

(2) Rau cov qauv uas muaj fiber ntau xws li nplej thiab nplej nplooj, cov nyiaj rho tawm yuav tsum raug txo kom tsim nyog, txwv tsis pub cov ntaub so ntswg sib tsoo thiab lysis yuav tsis ua kom tiav, ua rau cov txiaj ntsig qis ntawm RNA rho tawm.

(3) Rau cov ntaub so ntswg uas muaj dej ntau, xws li cov txiv hmab txiv ntoo pomegranate, txiv tsawb, txiv duaj txiv duaj, thiab lwm yam, cov qauv loj yuav tsum tau nce kom tsim nyog (100-200 mg yog xaiv tau).

(4) Cov ntaub so ntswg, xws li cov nplooj ntoo, rhizomes, cov txiv hmab txiv ntoo tawv thiab lwm yam ntaub ntawv feem ntau pom zoo kom siv cov kua nitrogen kom huv si mortar cov khoom xyaw rau hauv ib lub mortar, thiab tom qab ntawd mus rau theem extraction.Cov ntaub so ntswg homogenizers yuav tsis zoo rau homogenizing cov ntaub so ntswg, thiab feem ntau tsis pom zoo.

5. Kev ceev faj rau kev rho tawm RNA

(1) Cov qauv ntaub so ntswg yuav tsum yog tshiab li sai tau kom tsis txhob rov khov thiab thawing.

(2) Cov ntaub so ntswg yuav tsum tau muab tso rau hauv av thaum lub sij hawm rho tawm, thiab cov ntaub so ntswg yuav tsum tsis txhob tsawg dhau, cia nyob ib leeg ntau dhau.

(3) Lub sijhawm incubation txaus yuav tsum tau muab tom qab ntxiv cov lysate kom tag nrho lyse tus qauv.

(4) Thaum siv txoj kev Trizol rau extraction, lub hauv paus ntsiab lus ntawm absorbing lub supernatant tom qab stratification yog "xav kom nqus tau tsawg tshaj inhales ntau", thiab yuav tsum tsis txhob muab rho tawm mus rau nruab nrab txheej, txwv tsis pub nws yuav ua rau loj genomic DNA paug.

(5) Thaum ntxuav, cov kua ntxhua yuav tsum tau nkag mus rau hauv ib ncig ntawm lub raj phab ntsa kom huv si ntxuav.

(6) Rau txoj kev tshem tawm cov kab ke, ntxiv rau kev tshem tawm cov kab tom qab ntxuav, cov kab adsorption yuav tsum tau muab tso rau hauv lub rooj ntev zaum ultra-clean thiab tshuab rau 5-10 feeb kom tag nrho cov kuab tshuaj organic kom qhuav.

(7) Thaum kawg elution ntawm txoj kev kem, tom qab ntxiv DEPC dej, nws yuav tsum tau incubated rau 3-5 feeb, los yog cov dej DEPC yuav tsum tau rhuab mus rau 60 ° C ua ntej mus nce lub elution yield.Nyob rau hauv cov tsoos Trizol cleavage thiab isopropanol nag lossis daus txoj kev, qhov kawg RNA yog yaj nyob rau hauv DEPC dej, yog li lub sij hawm tsim nyog yuav tsum tau muab rau dissolution, thiab hauv qab ntawm lub centrifuge raj yuav tsum tsis tu ncua tshuab nrog ib tug pipette taub hau.

3 Three Ua rau thiab kev daws teeb meem rau qis RNA concentration / tsis zoo
 
1. Cov qoob loo qis dhau lawm
Cov qauv rho tawm tsawg dhau, tag nrho cov nyiaj tsis txaus, lossis cov qauv rho tawm ntau dhau thiab cov lysis tsis tiav;cov ntaub so ntswg lossis cov hlwb uas tsim nyog yuav tsum tau siv rau kev rho tawm, kev kho ua ntej ntawm cov qauv yuav tsum ua kom zoo, thiab cov lysis yuav tsum txaus.
 
2. Genome residues
Thaum rho tawm los ntawm Trizol txoj kev, thaum lub supernatant nqus mus rau hauv nruab nrab txheej tom qab txheej, loj genome paug yuav tshwm sim;yuav tsum tau saib xyuas ntxiv thaum txheej txheej kom tsis txhob nqus mus rau hauv nruab nrab txheej.Yog tias txoj kev kem siv rau kev rho tawm, cov khoom siv uas muaj DNase Kuv tuaj yeem raug xaiv rau kev rho tawm.Cov nucleic acid adsorbed ntawm daim nyias nyias yog ncaj qha zom nrog DNase I, uas tuaj yeem txo cov DNA seem.
 
3. RNA degradation
Nws yuav yog qhov degradation ntawm cov qauv rho tawm nws tus kheej, lossis qhov degradation tshwm sim thaum lub sij hawm rho tawm;kom deb li deb tau, cov qauv tshiab yuav tsum tau siv rau RNA rho tawm, thiab cov qauv sau tau yuav tsum muab cia rau hauv cov kua nitrogen los yog -80 ° C lub tub yees nyob rau lub sijhawm, thiab rov ua kom khov thiab thawing yuav tsum zam.RNase / DNase cov lus qhia pub dawb, cov raj centrifuge thiab lwm yam ntaub ntawv yuav tsum tau siv rau hauv RNA extraction txheej txheem.Cov txheej txheem rho tawm yuav tsum ceev li sai tau.RNA rho tawm yuav tsum tau muab tso rau hauv lub thawv dej khov thiab khaws cia ntawm -80 hauv lub sijhawm.Yog tias cov RNA rho tawm yuav tsum tau kuaj pom los ntawm gel electrophoresis, electrophoresis yuav tsum tau ua tam sim tom qab rho tawm, thiab cov electrophoresis tsis yuav tsum tau hloov nrog ib qho tshiab npaj.
 
4. Cov ntsev thiab cov organic solvent residues
Cov extraction reagents muaj phenol thiab guanidine ntsev, thiab cov tshuaj ntxuav tais diav muaj ethanol.Thaum lub sij hawm cov txheej txheem extraction, lysate tsis tau absorbed tag thiab muab pov tseg, thiab cov tshuaj ntxuav tsis tau qhuav tag.Cov ntsev seem thiab cov kuab tshuaj organic muaj teeb meem rau kev rov qab rov qab thiab PCR.Cov qib sib txawv ntawm inhibition, yog li cov ntaub so ntswg lysate yuav tsum tau muab tshem tawm tag nrho thaum lub sij hawm tshem tawm cov txheej txheem, thiab ntxuav yuav tsum txaus kom cov phab ntsa ntawm lub raj tuaj yeem ntxuav.Tsis tas li ntawd, lub raj yog emptied thiab tshuab yog ib kauj ruam tsim nyog, uas yuav ntxiv txo cov seem ntawm cov organic teeb meem.
 
Yog xav paub ntxiv txog RNA extraction, thov ua raws li peb lub vev xaib:
www.foreivd.com yog xav paub ntxiv.

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Post lub sij hawm: Dec-01-2022