Tungamira

Yakareba isina kukodha RNA, lncRNA isiri-coding RNA ine hurefu hunodarika mazana maviri nucleotides, kazhinji pakati pe200-100000 nt.lncRNA inodzora gene expression pa epigenetic, transcription uye post-transcriptional mazinga, uye inotora chikamu mu X chromosome kunyarara, genome imprinting uye chromatin modification, transcription activation, transcription interference, kutakura kwenyukireya, cell cycle regulation, cell differentiation regulation, uye dose compensation akakosha mhedzisiro, dosa remuripo mamwe maitiro akakosha. kudzivirira zvirwere zvevanhu, uye parizvino ndeimwe yenzvimbo dzinopisa mumunda webiomedicine.

01  Mhando uye maitiro eLncRNA

lncRNA yakakamurwa kuva mhando dzakawanda maererano nekwakasiyana siyana kwekuumbwa kwayo, sezvakaratidzwa mumufananidzo uri pamusoro.Ine zvinotevera maitiro:

1. lncRNAs anowanzo kureba, ane simba rekutaura uye nzira dzakasiyana dzekubatanidza panguva yekusiyanisa.
2. Kuenzaniswa nemajini ekukodha, lncRNA kazhinji ine mwero wakaderera wekutaura
3. Mazhinji lncRNAs ane pachena yenguva uye spatial kutaura zvakananga mukuita kwekusiyanisa kwematishu uye kukura.
4. Kune maitiro ekutaura mumamota nezvimwe zvirwere
5. Nzvimbo dze subcellular dzelncRNA dzakasiyana
6. Kutevedzana kwakaderera mukuchengetedza, asi basa rine imwe chiyero chekuchengetedza, nezvimwewo.

02 Mibvunzo Inowanzo bvunzwa nezve LncRNA Reverse Transcription

Nekuda kweiyo yakaderera yemukati yelncRNA mumaseru, kureba uye yakakwirira-yepamusoro chimiro, yakadaro RNA inowanzova neyakaomarara zvimiro senge stem loops kana hairpins, uye RT-qPCR inotarisana nezvinetso zvekusabudirira reverse transcription kana kushomeka kwekuita.

03 LncRNA reverse transcription solution

Mumutemo wepakati, maitiro eRNA mavhairasi achishandisa yavo reverse transcriptase kugadzira DNA vachishandisa RNA setemplate inonzi reverse transcription.Maitiro ekushandisa RNA virus reverse transcriptase kugadzira cDNA template in vitro inonzi reverse transcription.Zvinhu zvikuru zvinosanganisirwa mune reverse transcription system: RNA template, reverse transcriptase, primers uye zvimwe zvikamu.

1.RNA template

Iyo pesvedzero yeRNA template pane reverse transcription inoratidzwa muchimiro chayo, kuchena uye kuvimbika.Iyo yakakwirira kuchena uye kutendeseka kweRNA yakabviswa, ndiko kukwidziridza kugona kwereverse transcription uye kunowedzera kwakaringana mhedzisiro yehuwandu hunotevera.Iyo RNA yakacheneswa nezvinowanzo shandiswa RNA yekubvisa reagents kazhinji ine zvisaririra zvedoro uye guanidine munyu, izvo zvine simba inhibitory mhedzisiro pane yakawanda reverse transcriptase.

2.Reverse transcriptase

Reverse transcriptase ine simba guru mune yese reverse transcription system.Tembiricha yakakodzera reverse transcriptase inoshandiswa mumarabhoritari mazhinji inowanzoita 42°C.Muchokwadi, kuvhurwa kweiyo yepamusoro-level RNA chimiro kunoda tembiricha yepamusoro, uye zvinodikanwa zvereverse transcriptase zvakakwirira.

3.Primer

Reverse transcription primers zvinosanganisira gene-specific primers, random primers uye Oligo dT primers.Mazhinji malncRNA haana miswe yepolyA, uye zvakakosha kuti uenzanise zvakangoitika zvekutanga neOligo dT.

4.Zvimwe zvikamu

Tichifunga nezve chokwadi chekuti RNA inonyanya kushatisa, kuiswa kwezvikamu mune reverse transcription system kunogona kudzikiswa, izvo zvinogona kudzivirira zvinobudirira kupinda kweRNase uye kuve nechokwadi kufambira mberi kwakatsetseka kwereverse transcription.

Foregeneinowana kubva kuRNA template, reverse transcription kuqPCR

Nyatsoperekedza lncRNA yako kuona

Mhuka Yese RNA IsolationKit (ine gDNA yekubvisa column)

Sero Yese RNA IsolationKit (ine gDNA yekubvisa column)

Lnc-RT Hero TM I(Ne gDNase)(Super Premix yekutanga-strand cDNA synthesis kubva kulncRNA)

Real Time PCR EasyTM-SYBR Green I

Azvakanakira ofRNA kubviswa:

Iyo kit inoshandisa yechitatu-chizvarwa RNA yekubvisa tekinoroji, hapana chikonzero chekushandisa DNase.

11 maminetsi kubva 96, 24, 12, uye 6-chakanaka plate cultured masero, yakakwirira-inoshanda kutorwa kwegDNA-isina, yakakwirira-kuchena, yakakwirira-yemhando yesero RNA.

Zvakanakira zveLncRNA reverse transcription:

Reverse transcription system yakanyatso kugadzirirwa LncRNA kukurumidza kubvisa genomic DNA kusvibiswa.

Reverse transcription system ine kugadzikana kwemafuta uye ichine yakanaka reverse transcription performance pa50°C.

Matemplate ane yakakwira GC zvemukati uye yakaoma yechipiri chimiro inogona kudzoserwa nehunyanzvi hwepamusoro.

Zvakanakiraof qPCR:

Kupisa kutanga Foregene Taq Polymerase ine kukwirira kukwidziridza kunyatsoita, yakakwirira amplification senitivity, uye yakakwirira amplification chaiyo.

Iyo Yakagadziridzwa Real PCR Easy Mix inoita SYBR Green Ndine yakakwirira yekuona senitivity, uye fluorescence yayo yakanyanya 3-5 nguva yezvigadzirwa zvakafanana, izvo zvinogona kusangana nezvinodiwa zvemhando dzakasiyana dzefluorescence quantitative experiments.