Kutsogolera

RNA yayitali yosalemba zilembo, lncRNA ndi RNA yosalemba zilembo yokhala ndi utali wopitilira ma nucleotide 200, nthawi zambiri pakati pa 200-100000 nt.lncRNA imayang'anira mawonekedwe a jini pa epigenetic, transcript and post-transcriptal, ndipo imatenga nawo gawo pakuletsa kwa X chromosome, kusindikiza kwa genome ndi kusinthidwa kwa chromatin, kutsegulira kwa zolembera, kusokoneza zolembera, zoyendera za nyukiliya, malamulo ozungulira ma cell, kusiyanitsa kwa ma cell, ndi kubwezeredwa kwazinthu zina zofunika kwambiri (Dosa) kupewa matenda a anthu, ndipo pakali pano ndi amodzi mwa malo otentha kwambiri pankhani ya biomedicine.

01  Mitundu ndi mawonekedwe a LncRNA

lncRNA imagawidwa m'mitundu yambiri malinga ndi magwero osiyanasiyana a mapangidwe ake, monga momwe tawonetsera pa chithunzi pamwambapa.Lili ndi izi:

1. LncRNAs nthawi zambiri amakhala aatali, okhala ndi mawu osunthika komanso njira zolumikizirana zosiyanasiyana panthawi yosiyanitsa.
2. Poyerekeza ndi ma codec gene, lncRNA nthawi zambiri imakhala ndi mawu otsika
3. Ma lncRNA ambiri ali ndi mawonekedwe owoneka bwino akanthawi komanso apakati pakupanga kusiyanasiyana kwa minofu ndi chitukuko.
4. Pali khalidwe mawu zotupa ndi matenda ena
5. Malo a subcellular a lncRNA ndi osiyanasiyana
6. Zotsatizanazi ndizochepa posungirako, koma ntchitoyo ili ndi mlingo wina wotetezera, ndi zina zotero.

02 Mafunso Ofunsidwa Kawirikawiri Okhudza LncRNA Reverse Transcription

Chifukwa cha kuchepa kwa lncRNA m'maselo, kutalika kwautali ndi mawonekedwe apamwamba, RNA yotereyi nthawi zambiri imakhala ndi zida zovuta monga tsinde loops kapena ma hairpins, ndipo RT-qPCR imakonda kukhala ndi mavuto olembetsera reverse transcript kapena kuchepa kwachangu.

03 LncRNA reverse transcript solution

Mu ulamuliro wapakati, ndondomeko ya RNA mavairasi ntchito n'zosiyana transcriptase kuti lithe DNA ntchito RNA monga Chinsinsi amatchedwa m'mbuyo transcript.Njira yogwiritsira ntchito RNA virus reverse transcriptase kupanga cDNA template mu vitro imatchedwa reverse transcript.Mfundo zazikuluzikulu zomwe zikuphatikizidwa mu dongosolo la reverse transcript: RNA template, reverse transcriptase, primers ndi zigawo zina.

Chithunzi cha 1.RNA

Chikoka cha template ya RNA pakulemba mobwerera kumbuyo kumawonekera mu kapangidwe kake, chiyero ndi kukhulupirika.Kukwera kwa chiyero ndi kukhulupirika kwa RNA yotengedwa, kumapangitsa kuti zolembera zobwerera kumbuyo zikhale zolondola komanso zolondola kwambiri zotsatira zotsatila.The RNA woyeretsedwa ndi ambiri ntchito RNA m'zigawo reagents nthawi zambiri amakhala zotsalira za mowa ndi guanidine mchere, amene amphamvu chopinga zimakhudza kwambiri reverse transcriptase.

2.Reverse transcriptase

Reverse transcriptase ili ndi chikoka chachikulu pamakina onse am'mbuyo.Kutentha koyenera kwa reverse transcriptase yomwe imagwiritsidwa ntchito m'ma laboratories ambiri nthawi zambiri ndi 42°C.Ndipotu, kutsegulidwa kwa mapangidwe apamwamba a RNA kumafuna kutentha kwakukulu, ndipo zofunikira za reverse transcriptase ndizokwera.

3.Choyamba

Zoyambira zosinthira zimaphatikiza zoyambira zamtundu wamtundu, zoyambira mwachisawawa ndi zoyambira za Oligo dT.Ma lncRNA ambiri alibe michira ya polyA, ndipo ndikofunikira kufananiza zoyambira mwachisawawa ndi Oligo dT.

4.Zigawo zina

Poganizira kuti RNA ndiyosavuta kunyonyotsoka, kuyambitsa kwa zigawo za reverse transcript kutha kuchepetsedwa, zomwe zingalepheretse kulowa kwa RNase ndikuwonetsetsa kuti zolembedwazo zikuyenda bwino.

Foregeneamalandira kuchokera ku template ya RNA, sinthani zolembera kupita ku qPCR

Tsatirani mokwanira kuzindikira kwanu kwa lncRNA

Animal Total RNA ImtendereKit (yokhala ndi gawo lochotsa gDNA)

Cell Total RNA ImtendereKit (yokhala ndi gawo lochotsa gDNA)

Lnc-RT HeroTM I(Ndi gDNase)(Super Premix for first-strand cDNA synthesis from lncRNA)

Real Time PCR EasyTM-SYBR Green I

Aubwino ofKutulutsa kwa RNA:

Chidacho chimagwiritsa ntchito ukadaulo wa m'badwo wachitatu wa RNA, osafunikira kugwiritsa ntchito DNase.

Mphindi 11 kuchokera ku 96, 24, 12, ndi 6-ma cell otukuka bwino mbale, kutulutsa kwapamwamba kwa gDNA-free, chiyero chapamwamba, ma cell a RNA apamwamba kwambiri.

Ubwino wa lncRNA reverse transcript:

Dongosolo la reverse transcript lomwe linapangidwira kuti LncRNA lichotse mwachangu kuipitsidwa kwa DNA.

Makina osindikizira obwerera m'mbuyo amakhala ndi kukhazikika kwa kutentha kwambiri ndipo amakhalabe ndi ntchito yabwino yosinthira pa 50 ° C.

Ma templates okhala ndi GC yayikulu komanso mawonekedwe achiwiri ovuta amatha kusinthidwa ndikuchita bwino kwambiri.

Ubwino wakeof qPCR:

Kuyamba kotentha kwa Foregene Taq Polymerase kumakhala ndi mphamvu zokulirapo, kukhudzika kwamphamvu, komanso kumveketsa bwino kwambiri.

Kusakaniza kwa Real PCR Easy Mix kumapangitsa SYBR Green I kukhala ndi chidziwitso chapamwamba chodziwikiratu, ndipo mphamvu yake ya fluorescence ndi 3-5 nthawi ya zinthu zofanana, zomwe zingathe kukwaniritsa zosowa za mitundu yosiyanasiyana ya kuyesa kwamtundu wa fluorescence.