Taw qhia:

RNA, lub npe scientific ribonucleic acid, nws cov ntawv luv los ntawm RiboNucleic Acid, yog ib hom nucleic acid tsim los ntawm kev sib txuas tsawg kawg kaum ob ntawm ribonucleotides los ntawm phosphodiester bonds.

Lub hauv paus ntsiab lus ntawm kev tshawb nrhiav RNA yog txhawm rau rho tawm cov pa phem tag nrho RNA, thiab lub hauv paus ntsiab lus ntawm kev rho tawm RNA tuaj yeem ua tau raws li hauv qab no:

1. cuam tshuam cov hlwb ua ntej

2. Tom qab ntawd tshem tawm macromolecular impurities (proteins, lipids, DNA, thiab lwm yam)

3. Thaum kawg, RNA yog precipitated thaum tshem cov ntsev thiab cov organic solvents, thiab ntxiv purifying RNA.

Yav dhau los thiab tam sim no ntawm kev rho tawm txoj kev

Thawj tiam ntawm RNA extraction method yog TRIzol los nag txoj kev (reagents siv xws li guanidine isothiocyanate, phenol, chloroform, thiab lwm yam) tsim los ntawm ib tug yawg npe hu ua Piotr Chomczynski thiab pog Nicoletta Sacchi, vim tias qhov kev tsim no tso cai rau RNA rho tawm sai thiab yooj yim, txij li thaum ntawd pib qhov kev tshawb fawb.

Tom qab ntawd, yawg tau muag daim ntawv pov thawj no rau Invitrogen, thiab Invitrogen tau txhawb nqa txoj kev no rau lub ntiaj teb thiab dhau los ua txoj kev siv dav.

Tom qab ntawd, Invitrogen tau txais los ntawm Thermo!

Cov Khoom Muag Khoom: Invitrogen's TRIzol reagent

Txawm li cas los xij, txoj kev nag lossis daus TRIzol yuav tsum muaj ntau yam nag lossis daus thiab rho tawm ntawm RNA, thiab cov reagents siv yog tshuaj lom thiab siv sijhawm ntev los ua haujlwm.

Yog li ntawd, cov txheej txheem thib ob tiam-enzyme purification txoj kev tau los ua, siv silica gel membrane raws li ib qho tshwj xeeb nucleic acid adsorption cov ntaub ntawv kom ua kom rov zoo ntawm RNA hauv cov qauv thaum tshem tawm lwm yam impurities.

Txoj kev thib ob ua kom yooj yim rau kev ua haujlwm raws li kev ua haujlwm ruaj khov, txo qhov kev pheej hmoo, thiab txhim kho kev ua haujlwm ntawm RNA extraction.

Cov khoom lag luam sawv cev: QIAGEN RNeasy thiab feem ntau cov khoom siv hauv tsev

Txawm li cas los xij, ob qho tib si thawj thiab thib ob txoj hauv kev xav tau kev siv DNase kom tshem tawm cov kab mob DNA, uas ua rau qee qhov tsis yooj yim rau cov kev sim tom ntej.Residual DNase yuav txo qhov kev ua tau zoo ntawm kev rov qab rov qab thiab txo qis thawj-strand cDNA synthesis, yog li cuam tshuam rau qhov tseeb ntawm cov txiaj ntsig.

Yog li ntawd, lub thib peb tiam extraction txoj kev yog optimized nyob rau hauv lub hauv paus ntawm lub thib ob tiam.Qhov kev sim tsis tas yuav ntxiv cov enzymes, thiab RNA tuaj yeem yooj yim tau nrog tsuas yog ob kab.

Kem 1: DNA-Tshuaj Kem kom tshem DNA

Kem 2: RNA nkaus xwb Kem tshwj xeeb adsorbs thiab rov qab RNA

Xws li kev ua haujlwm yooj yim tsis tsuas yog txuag lub sijhawm, tab sis kuj tseem pab txhawb kev ua haujlwm ntawm cov kev sim hauv qab!

Cov khoom lag luam sawv cev: QIAGEN RNeasy Plus thiabForegeneRNA purification series khoom

(RNeasy Plus)

(Foregene covPlant Total RNA Isolation Kit)

Qhia rau koj ntsiag to!Foregene's RNA cov khoom siv kuj nthuav tawm qhov yooj yim-rau-siv RNase Eraser, uas tuaj yeem tshem tawm tag nrho 400μg ntawm RNase ntawm cov khoom nto hauv 1 feeb, tsis tas yuav ntxiv cov tshuaj lom rau tib neeg lub cev!

Duab1: Kev sib piv cov txheej txheem ua haujlwm

Foregene cov khoom tuaj yeem ua tiav RNA rho tawm hauv ob zaj nkauj xwb, tsis txhob ua rau lwm tus khib ntawm koj qhov kev sim ua tiav!

Duab2:Experimental cov txiaj ntsig daim duab sib piv

Thawj tiam (1: TRIzol)

Qhov thib ob tiam (2: RNeasy ntawm QIAGEN)

Peb tiam (3: QIAGEN's RNeasy plus, 4: Foregene)

Duab3: qPCR graph (amplification curve graph)

Thawj tiam (ntsuab txhais tau tias TRIzol -, liab txhais tau tias TRIzol +)

Lub tiam thib ob (lub ntuj xiav txhais tau tias QIAGEN RNeasy-, liab txhais tau tias QIAGEN RNeasy +)

Peb tiam (xiav txhais tau tias QIAGEN RNeasy ntxiv, ntsuab txhais tau tias Foregene)

(Ceeb Toom: qPCR graph "-" qhia tias DNase tsis tau ntxiv thiab DNA tsis tau raug tshem tawm. Yog li, CT siab dua thiab qhov tshwm sim tsis raug; "+" qhia tias DNase tau ntxiv thiab DNA kis tau raug tshem tawm, qhov tshwm sim yog qhov tseeb)

Txhua qhov kev hloov kho tshiab txhais tau hais tias kev ua haujlwm ntawm qhov kev sim yog tsim nyob rau hauv ib qho yooj yim thiab ntau tus neeg siv-phooj ywg kev taw qhia.

Foregene lub hom phiaj los pab cov kws tshawb fawb tshawb fawb ntau dua txuag lub sijhawm sim thiab tus nqi, tau txais cov txiaj ntsig kev sim siab, thiab sib koom ua ke tsim cov txiaj ntsig kev tshawb fawb muaj txiaj ntsig zoo dua.