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Ntev tsis-coding RNA, lncRNA yog ib qho tsis-coding RNA nrog ntev dua 200 nucleotides, feem ntau ntawm 200-100000 nt.lncRNA tswj gene qhia ntawm epigenetic, transcription thiab post-transcriptional theem, thiab koom nyob rau hauv X chromosome silencing, genome imprinting thiab chromatin hloov, transcription activation, transcription interference, nuclear thauj, cell voj voog kev tswj, cell sib txawv kev cai, thiab koob tshuaj them nyiaj (Cov tshuaj tiv thaiv kab mob cuam tshuam rau tib neeg), thiab ntau lwm yam tseem ceeb tshwm sim. tam sim no yog ib qho chaw kub kub hauv kev lag luam biomedicine.

01  Hom thiab yam ntxwv ntawm LncRNA

lncRNA muab faib ua ntau hom raws li qhov sib txawv ntawm nws qhov tsim, raws li qhia hauv daim duab saum toj no.Nws muaj cov yam ntxwv hauv qab no:

1. lncRNAs feem ntau yog ntev dua, nrog dynamic qhia thiab txawv splicing txoj kev thaum sib txawv
2. Muab piv nrog cov noob caj noob ces, lncRNA feem ntau muaj cov lus qhia qis dua
3. Feem ntau lncRNAs muaj qhov pom tseeb ntawm sab cev nqaij daim tawv thiab spatial qhia tshwj xeeb hauv cov txheej txheem ntawm cov ntaub so ntswg sib txawv thiab kev loj hlob
4. Muaj cov yam ntxwv ntawm cov qog thiab lwm yam kab mob
5. Qhov chaw subcellular ntawm lncRNA muaj ntau haiv neeg
6. Cov kab ke tsis tshua muaj kev txuag, tab sis kev ua haujlwm muaj qee qhov kev txuag, thiab lwm yam.

02 Cov lus nug nquag nug txog LncRNA Reverse Transcription

Vim tias cov ntsiab lus qis ntawm lncRNA hauv cov hlwb, ntev ntev thiab cov qauv siab, xws li RNA feem ntau muaj cov txheej txheem nyuaj xws li qia loops lossis hairpins, thiab RT-qPCR yog ib qho teeb meem ntawm kev ua tsis tiav rov qab los yog ua haujlwm tsis zoo.

03 LncRNA thim rov qab kev daws teeb meem

Hauv kev tswj hwm hauv nruab nrab, cov txheej txheem ntawm RNA cov kab mob siv lawv tus kheej rov qab transcriptase los ua ke DNA siv RNA ua tus qauv hu ua thim rov qab.Cov txheej txheem ntawm kev siv RNA tus kab mob thim rov qab transcriptase los ua ke cDNA template hauv vitro hu ua thim rov qab.Cov ntsiab lus tseem ceeb muaj nyob rau hauv qhov kev hloov pauv rov qab: RNA template, thim rov qab transcriptase, primers thiab lwm yam khoom.

1.RNA template

Kev cuam tshuam ntawm RNA template ntawm kev hloov pauv rov qab yog tshwm sim hauv nws cov qauv, purity thiab kev ncaj ncees.Qhov siab dua qhov purity thiab kev ncaj ncees ntawm cov rho tawm RNA, qhov ua tau zoo ntawm kev thim rov qab ntau dua thiab qhov tseeb dua cov txiaj ntsig tom qab.RNA purified los ntawm feem ntau siv RNA extraction reagents feem ntau muaj residues ntawm cawv thiab guanidine ntsev, uas muaj ib tug muaj zog inhibitory nyhuv ntawm feem ntau reverse transcriptase.

2.Reverse transcriptase

Rov qab transcriptase muaj qhov cuam tshuam tseem ceeb hauv tag nrho cov kev hloov pauv rov qab.Qhov ntsuas kub tsim nyog rau qhov rov qab transcriptase siv nyob rau hauv ntau lub chaw soj nstuam yog feem ntau 42 ° C.Qhov tseeb, qhov qhib ntawm cov qauv RNA qib siab yuav tsum muaj qhov kub thiab txias dua, thiab qhov yuav tsum tau ua rau qhov rov qab transcriptase yog siab dua.

3.Primer

Rov qab cov ntaub ntawv rov qab suav nrog cov noob tshwj xeeb primers, random primers thiab Oligo dT primers.Feem ntau lncRNAs tsis muaj polyA tails, thiab nws yog ib qho tseem ceeb uas yuav tsum tau ua kom zoo sib xws cov random primers nrog Oligo dT.

4.Lwm yam khoom

Raws li qhov tseeb hais tias RNA yog qhov tshwj xeeb tshaj yog yooj yim rau degrade, cov lus qhia ntawm cov khoom nyob rau hauv qhov rov qab transcription system tuaj yeem txo qis, uas tuaj yeem tiv thaiv kev nkag ntawm RNase thiab xyuas kom meej qhov kev vam meej ntawm kev rov qab rov qab.

Fcov oregenetau los ntawm RNA template, thim rov qab mus rau qPCR

Ua tiav escort koj lncRNA nrhiav pom

Tsiaj Tag Nrho RNA Ikev kaj siabCov khoom siv (nrog gDNA tshem tawm kem)

Cell Total RNA Ikev kaj siabCov khoom siv (nrog gDNA tshem tawm kem)

Lnc-RT HeroTM I (Nrog gDNase) (Super Premix rau thawj-strand cDNA synthesis los ntawm lncRNA)

Real Time PCR EasyTM-SYBR Green I

Aqhov zoo ofRNA rho tawm:

Cov khoom siv siv thev naus laus zis RNA thib peb, tsis tas yuav siv DNase.

11 feeb los ntawm 96, 24, 12, thiab 6-zoo phaj kab lis kev cai, kev ua haujlwm zoo ntawm gDNA-dawb, siab-purity, zoo tagnrho cell RNA.

Qhov zoo ntawm lncRNA reverse transcription:

Ib qho kev hloov pauv rov qab tshwj xeeb tsim rau LncRNA kom tshem tawm cov kab mob DNA sai sai.

Qhov rov qab transcription system muaj kev ruaj ntseg thermal thiab tseem muaj qhov zoo rov qab hloov pauv ntawm 50 ° C.

Templates nrog siab GC cov ntsiab lus thiab cov txheej txheem thib ob nyuaj tuaj yeem thim rov qab nrog kev ua haujlwm siab.

Qhov zooof qPCR:

Kub pib Foregene Taq Polymerase muaj ntau dua amplification efficiency, siab dua amplification rhiab heev, thiab ntau dua amplification tshwj xeeb

Qhov optimized Real PCR Yooj Yim Mix ua rau SYBR Green Kuv muaj kev tshawb pom ntau dua, thiab nws qhov kev siv fluorescence yog 3-5 npaug ntawm cov khoom zoo sib xws, uas tuaj yeem ua tau raws li qhov xav tau ntawm ntau hom fluorescence quantitative thwmsim.