Jagoranci

Dogon RNA mara coding, lncRNA RNA ce mara coding mai tsayi sama da 200 nucleotides, gabaɗaya tsakanin 200-100000 nt.lncRNA yana daidaita maganganun kwayoyin halitta a epigenetic, rubutun rubutu da matakan bayan bayanan, kuma yana shiga cikin shiru na X chromosome, genome imprinting da gyaran chromatin, kunna rubutun, tsangwama rubutun, jigilar nukiliya, tsarin sake zagayowar tantanin halitta, tsarin bambance-bambancen tantanin halitta, da kuma tasirin sakamako na ramuwa da yawa (mahimmancin sakamako na ramuwa na ɗan adam) kuma a halin yanzu suna ɗaya daga cikin wurare masu zafi a fagen nazarin halittu.

01  Nau'i da halaye na LncRNA

An raba lncRNA zuwa nau'i-nau'i da yawa bisa ga mabanbanta tushen samuwarsa, kamar yadda aka nuna a cikin adadi na sama.Yana da halaye masu zuwa:

1. lncRNAs yawanci sun fi tsayi, tare da magana mai ƙarfi da hanyoyin rarraba daban-daban yayin rarrabuwa
2. Idan aka kwatanta da codeing genes, lncRNA yawanci yana da ƙananan matakin magana
3. Yawancin lncRNA suna da ƙayyadaddun magana na ɗan lokaci da sarari a cikin tsarin rarrabuwar nama da haɓakawa.
4. Akwai maganganu masu ma'ana a cikin ciwace-ciwacen daji da sauran cututtuka
5. Wuraren subcellular na lncRNA sun bambanta
6. Jerin yana da ƙasa a cikin kiyayewa, amma aikin yana da takamaiman matakin kiyayewa, da dai sauransu.

02 Tambayoyin da ake yawan yi game da Rubutun LncRNA

Saboda ƙarancin abun ciki na lncRNA a cikin sel, tsayin tsayi da babban tsari, irin wannan RNA sau da yawa yana da hadaddun sifofi kamar madaukai masu tushe ko gashin gashi, kuma RT-qPCR yana fuskantar matsalolin rashin nasarar rubutun juyi ko ƙarancin inganci.

03 Maganin juyar da rubutun LncRNA

A cikin ka'ida ta tsakiya, tsarin ƙwayoyin cuta na RNA suna amfani da nasu juzu'i na juzu'i don haɗa DNA ta amfani da RNA azaman samfuri ana kiransa reverse transcription.Tsarin amfani da kwayar cutar ta RNA mai juyo bayanan rubutu don haɗa samfuri na cDNA a cikin vitro ana kiransa reverse transcription.Babban abubuwan da aka haɗa a cikin tsarin jujjuyawar baya: samfuri na RNA, juzu'i na jujjuyawar, firamare da sauran abubuwan haɗin gwiwa.

1. RNA samfuri

Tasirin samfurin RNA akan juyar da rubutu yana bayyana a tsarinsa, tsarkinsa da amincinsa.Mafi girman tsarki da mutuncin RNA da aka fitar, mafi girman ingancin juzu'i da kuma ingantaccen sakamakon ƙididdigewa na gaba.RNA da aka tsarkake ta hanyar reagents na hakar RNA da aka saba amfani da su sau da yawa ya ƙunshi ragowar barasa da gishirin guanidine, waɗanda ke da tasirin hanawa mai ƙarfi akan mafi yawan juzu'i.

2. Reverse transcriptase

Reverse transcriptase yana da mahimmin tasiri a cikin gabaɗayan tsarin rubutun baya.Matsakaicin zafin da ya dace don jujjuya bayanan da aka yi amfani da shi a yawancin dakunan gwaje-gwaje shine gabaɗaya 42°C.A haƙiƙa, buɗe babban tsarin RNA yana buƙatar zafin jiki mafi girma, kuma abubuwan buƙatun juzu'i sun fi girma.

3.Firamare

Maƙallan rubutun juye-juye sun haɗa da ƙayyadaddun ƙayyadaddun ƙayyadaddun kwayoyin halitta, na'urorin bazuwar da na Oligo dT.Yawancin lncRNA ba su ƙunshi wutsiyar polyA ba, kuma yana da mahimmanci a daidaita daidaitattun abubuwan da aka bazu tare da Oligo dT.

4.Sauran abubuwa

Dangane da gaskiyar cewa RNA yana da sauƙin ƙasƙantar da kai, ana iya rage ƙaddamar da abubuwan da ke cikin tsarin rubutun baya, wanda zai iya hana shigowar RNase yadda ya kamata da tabbatar da ingantaccen ci gaba na juzu'i.

Foregeneyana samuwa daga samfurin RNA, juyar da rubutu zuwa qPCR

Gabaɗaya rakiyar gano lncRNA naku

Jimlar Dabbobin RNA Ikwanciyar hankaliKit (tare da ginshiƙin cire gDNA)

Total Cell RNA Ikwanciyar hankaliKit (tare da ginshiƙin cire gDNA)

Lnc-RT HeroTM I (Tare da gDNase) (Super Premix don haɗin cDNA na farko daga lncRNA)

Real Time PCR EasyTM-SYBR Green I

Aabũbuwan amfãni ofHakar RNA:

Kit ɗin yana amfani da fasahar hakar RNA ta ƙarni na uku, babu buƙatar amfani da DNAse.

Minti 11 daga 96, 24, 12, da 6-rabo mai kyau farantin sel al'ada, ingantaccen haɓakar gDNA mara kyauta, tsafta mai inganci, jimlar tantanin halitta RNA.

Fa'idodin rubutun lncRNA:

Tsarin jujjuya bayanan da aka kirkira musamman don LncRNA don cire gurɓataccen DNA da sauri.

Tsarin rubutun juyi yana da babban kwanciyar hankali na zafi kuma har yanzu yana da kyakkyawan aikin juyi a 50°C.

Samfuran da ke da babban abun ciki na GC da hadadden tsari na sakandare na iya juyawa tare da babban inganci.

Amfaniof qPCR:

Farawa mai zafi Foregene Taq Polymerase yana da ingantaccen haɓakawa, haɓaka haɓakawa, da ƙayyadaddun haɓakawa mafi girma.

A gyara Real PCR Easy Mix sa SYBR Green Ina da mafi girma ganewa hankali, da kuma fluorescence tsanani ne 3-5 sau fiye da na irin wannan kayayyakin, wanda zai iya saduwa da bukatun daban-daban na fluorescence gwada yawa gwaje-gwaje.