Ke alakaʻi

ʻO RNA non-coding lōʻihi, ʻo lncRNA he RNA non-coding me ka lōʻihi ma mua o 200 nucleotides, maʻamau ma waena o 200-100000 nt.Hoʻoponopono ʻo lncRNA i ka hōʻike ʻana i ke ʻano ma ka epigenetic, transcription a me post-transcriptional pae, a komo i ka X chromosome silencing, genome imprinting a me ka hoʻololi chromatin, transcription activation, transcription interference, nuclear transport, cell cycle regulation, cell differentiation regulation, a me ka hopena o nā hopena pili (Dosage marence e pili ana i nā hopena pili pili kanaka) , a i kēia manawa kekahi o nā wahi wela ma ke kahua o ka biomedicine.

01  Nā ʻano a me nā ʻano o LncRNA

Ua māhele ʻia ka lncRNA i nā ʻano he nui e like me nā kumu like ʻole o kona hoʻokumu ʻana, e like me ka mea i hōʻike ʻia ma ke kiʻi ma luna.Loaʻa nā ʻano penei:

1. ʻOi aku ka lōʻihi o nā lncRNA, me ka hōʻike ikaika a me nā ʻano hana splicing ʻokoʻa i ka wā o ka ʻokoʻa
2. Hoʻohālikelike ʻia me nā genes coding, ʻoi aku ka haʻahaʻa o ka lncRNA
3. Loaʻa i ka hapa nui o nā lncRNA ke ʻano kikoʻī o ke kino a me ka spatial i ke kaʻina o ka ʻokoʻa a me ka hoʻomohala ʻana.
4. Aia kekahi mau hiʻohiʻona i nā ʻōpū a me nā maʻi ʻē aʻe
5. He ʻokoʻa nā wahi subcellular o lncRNA
6. He haʻahaʻa ke kaʻina i ka mālama ʻana, akā aia ka hana i kekahi degere o ka mālama ʻana, etc.

02 Nīnau pinepine e pili ana i ka LncRNA Reverse Transcription

Ma muli o ka haʻahaʻa haʻahaʻa o ka lncRNA i loko o nā pūnaewele, lōʻihi ka lōʻihi a me ke ʻano kiʻekiʻe, loaʻa pinepine ka RNA i nā hale paʻakikī e like me nā puka lou a i ʻole nā ​​hairpins, a hiki i ka RT-qPCR ke pilikia i nā pilikia o ka hoʻohuli ʻole ʻana a i ʻole ka hana haʻahaʻa.

03 LncRNA hoʻololi hoʻololi unuhi

Ma ka lula koʻikoʻi, ʻo ke kaʻina hana o nā maʻi virus RNA e hoʻohana ana i kā lākou iho transcriptase hoʻohuli e synthesize DNA me ka hoʻohana ʻana i ka RNA ma ke ʻano he ʻano i kapa ʻia ʻo reverse transcription.ʻO ke kaʻina hana o ka hoʻohana ʻana i ka RNA virus reverse transcriptase e synthesize cDNA template in vitro i kapa ʻia he reverse transcription.ʻO nā mea nui i hoʻokomo ʻia i loko o ka ʻōnaehana transcription reverse: RNA template, reverse transcriptase, primers a me nā mea ʻē aʻe.

1. RNA template

Hōʻike ʻia ka mana o ka template RNA i ka hoʻololi ʻana i kona ʻano, ka maʻemaʻe a me ka pololei.ʻOi aku ka kiʻekiʻe o ka maʻemaʻe a me ka pololei o ka RNA i unuhi ʻia, ʻoi aku ka kiʻekiʻe o ka pono o ka hoʻohuli ʻana a me ka pololei o nā hopena quantitative ma hope.ʻO ka RNA i hoʻomaʻemaʻe ʻia e ka RNA extraction reagents i hoʻohana pinepine ʻia i ke koena o ka waiʻona a me nā paʻakai guanidine, he hopena paʻa ikaika i ka hapa nui o ka transcriptase.

2. Reverse transcriptase

He mana koʻikoʻi ko Reverse transcriptase i ka ʻōnaehana hoʻololi hope.ʻO ka mahana kūpono no ka reverse transcriptase i hoʻohana ʻia ma ka hapa nui o nā laboratories ma ke ʻano he 42°C.ʻO ka ʻoiaʻiʻo, ʻo ka wehe ʻana o ka hoʻolālā RNA kiʻekiʻe e koi i kahi mahana kiʻekiʻe, a ʻoi aku ka kiʻekiʻe o nā koi no ka reverse transcriptase.

3.Kumuhana

Hoʻokomo ʻia nā kumu mua transcription hoʻihoʻi i nā kumu mua kikoʻī gene, nā kumu mua a me nā kumu mua Oligo dT.ʻAʻole i loaʻa i ka hapa nui o nā lncRNA nā huelo polyA, a he mea nui e hoʻohālikelike pono i nā primers random me Oligo dT.

4. Nā mea'ē aʻe

Ma muli o ka maʻalahi o ka hoʻohaʻahaʻa ʻana i ka RNA, hiki ke hoʻemi ʻia ka hoʻokomo ʻana o nā ʻāpana i ka ʻōnaehana transcription hope, hiki ke pale pono i ke komo ʻana o RNase a hōʻoia i ka holomua holomua o ka hoʻohuli ʻana.

Foregeneloaʻa mai ka RNA template, hoʻohuli transcription i qPCR

Hoʻopili piha i kāu ʻike lncRNA

Holoholona Huina RNA IhoʻomahaKiko (me ke kolamu wehe gDNA)

Pūnaehana Huina RNA IhoʻomahaKiko (me ke kolamu wehe gDNA)

Lnc-RT HeroTM I(Me gDNase)(Super Premix no ke kaula cDNA synthesis mua mai lncRNA)

ʻO ka manawa maoli PCR EasyTM-SYBR Green I

Anā mea maikaʻi ofRNA unuhi:

Hoʻohana ka kit i ka ʻenehana unuhi RNA ʻekolu, ʻaʻohe pono e hoʻohana i ka DNase.

11 mau minuke mai 96, 24, 12, a me 6-well plate cultured cell, kiʻekiʻe-efficiency extraction of gDNA-free, high-purity, high-quality total cell RNA.

ʻO nā pono o ka lncRNA reverse transcription:

ʻO kahi ʻōnaehana transcription hoʻohuli i hoʻomohala kūikawā ʻia no LncRNA e hoʻoneʻe koke i ka ʻino DNA genomic.

Loaʻa i ka ʻōnaehana hoʻololi hope ke kūpaʻa wela kiʻekiʻe a ʻoi aku ka maikaʻi o ka hana hoʻihoʻi ʻana ma 50°C.

Hiki ke hoʻohuli ʻia nā template me ka ʻike GC kiʻekiʻe a me ka hoʻolālā lua paʻakikī me ka hana kiʻekiʻe.

Ponoof qPCR:

ʻO ka hoʻomaka wela ʻo Foregene Taq Polymerase i ʻoi aku ka maikaʻi o ka amplification kiʻekiʻe, ʻoi aku ka nui o ka hoʻonui ʻana, a me nā kikoʻī hoʻonui kiʻekiʻe.

ʻO ka optimized Real PCR Easy Mix e hana i ka SYBR Green He kiʻekiʻe aʻe koʻu naʻau, a ʻo kona ikaika fluorescence he 3-5 mau manawa o nā huahana like, hiki ke hoʻokō i nā pono o nā ʻano like ʻole o ka fluorescence quantitative hoʻokolohua.