Ka lōʻihi non-coding sequencing-Illumina
Pono lawelawe
● Pono lawelawe
● ʻO ke kelepona a me ke kiko kiko
● Hōʻike a hōʻike ka pae kikoʻī i ka hoʻololi ʻana i ka manaʻo ikaika
● Nā hiʻohiʻona pololei o ka hōʻike manawa a me ka lewa
● Hoʻopili hui me ka ʻikepili mRNA.
● Hāʻawi i nā hualoaʻa ma muli o BMKCloud: Loaʻa ka ʻili ʻikepili i hana ʻia ma ka paepae.
● Pono nā lawelawe ma hope o ke kūʻai aku no 3 mahina ma hope o ka pau ʻana o ka papahana
Nā Koina Laʻana a me ka Hoʻouna ʻana
| Hale Waihona Puke | Papahana | Manaʻo ʻikepili | ʻIkepili QC |
| rRNA hoopau | Illumina PE150 | 10 Gb | Q30≥85% |
| Conc.(ng/μl) | Ka nui (μg) | Maemae | Pono pono |
| ≥ 100 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Hoʻopaʻa ʻia a ʻaʻole hoʻi i ka protein a i ʻole DNA contamination i hōʻike ʻia ma ka gel. | No nā mea kanu: RIN≥6.5; No nā holoholona: RIN≥7.0; 5.0≥28S/18S≥1.0; palena a ʻaʻole kiʻekiʻe kumu |
Nucleotides:
Tissue: Kaumaha (maloʻo): ≥1 g
*No ka ʻiʻo liʻiliʻi ma mua o 5 mg, manaʻo mākou e hoʻouna i kahi hāpana kikoo uila (i loko o ka nitrogen wai).
Hoʻokuʻu ʻia ke kelepona: helu kelepona = 3×107
* Manaʻo mākou e hoʻouna i ka lysate cell frozen.Inā ʻoi aku ka liʻiliʻi o kēlā cell ma mua o 5 × 105, ʻōlelo ʻia ka flash frozen i loko o ka wai nitrogen.
Nā laʻana koko:
PA×geneBloodRNATube;
6mLTRIzol a me 2mL koko(TRIzol:Blood=3:1)
Manaʻo ʻia ka hāʻawi laʻana
ʻO ka pahu: 2 ml centrifuge tube (ʻAʻole ʻōlelo ʻia ʻo Tin foil)
Laʻana lepili: Pūʻulu+hoʻopiʻi e laʻa me A1, A2, A3;B1, B2, B3... ...
Hoʻouna:
1.Dry-ice: Pono e hoʻopiliʻia nā laʻana i loko o nāʻeke a kanuʻia i loko o ka hau maloʻo.
2.RNAstable tubes: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA ma ka RNA stabilization tube (eg RNAstable®) a hoʻouna ʻia i loko o ka lumi wela.
Holo Hana Hana
Hoʻolālā hoʻokolohua
Hāʻawi laʻana
RNA unuhi
Kūkulu hale waihona puke
Ke kaʻina ʻana
ʻIkepili ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
Bioinformatics
1.LncRNA hoʻokaʻawale
ʻO LncRNA i wānana ʻia e nā polokalamu ʻehā ma luna nei ua hoʻokaʻawale ʻia i 4 mau ʻāpana: lincRNA, anti-sense-LncRNA, intronic-LncRNA;manaʻo-LncRNA.Ua hōʻike ʻia ka helu LncRNA ma ka histogram ma lalo nei.
Hoʻokaʻawale LncRNA
2.Cis-target genes o DE-lncRNA enrichment analysis
Ua hoʻohana ʻia ʻo ClusterProfiler ma ka loiloi hoʻonui ʻana o GO ma luna o nā genes cis-targeted o lncRNA (DE-lncRNA) i hōʻike ʻokoʻa ʻia, e pili ana i nā kaʻina hana olaola, nā hana molekala a me nā ʻāpana kelepona.ʻO ka loiloi hoʻonui GO kahi kaʻina e ʻike ai i nā huaʻōlelo GO i hoʻonui nui ʻia i alakaʻi ʻia e DEG i hoʻohālikelike ʻia me ka genome holoʻokoʻa.Ua hōʻike ʻia nā huaʻōlelo i hoʻonui ʻia ma ka histogram, bubble chart, etc.
ʻO nā genes Cis-targeted o DE-lncRNA enrichment analysis -Bubble chart
3. Ma ka hoohalike ana i ka loihi, ka helu exon, ORF a me ka huina hoike o mRNA a me lncRNA, hiki ia kakou ke hoomaopopo i ka like ole o ka hale, ke kaʻina a pela aku ma waena o ia mau mea, a e hooiaio no hoi ina ua like ka moolelo lncRNA i wanana ia e kakou me na ano nui.
Hihia BMK
Deregulated lncRNA hōʻike hōʻike i loko o ka ʻiole māmā adenocarcinomas me KRAS-G12D mutation a me P53 knockout
Paʻi ʻia:Journal of Cellular and Molecular Medicine，2019
Hoʻolālā kaʻina
ʻIlumina
ʻOhi laʻana
Ua loaʻa nā pūnaewele NONMMUT015812-knockdown KP (shRNA-2) a me ka mana maikaʻi ʻole (sh-Scr) i ka lā 6 o kahi maʻi viral kikoʻī.
Nā hopena koʻikoʻi
Ke noiʻi nei kēia haʻawina i nā lncRNA i hōʻike ʻole ʻia i loko o ka adenocarcinoma māmā ʻiole me P53 knockout a me ka mutation KrasG12D.
1.6424 lncRNA i hōʻike ʻokoʻa (≥ 2-fold hoʻololi, P <0.05).
2. Ma waena o nā 210 lncRNAs (FC≥8), 11 lncRNAs 'ōlelo ua hooponopono ia e P53, 33 lncRNAs e KRAS a me 13 lncRNAs e hypoxia i loko o ka mua KP pūnaewele, pakahi.
3.NONMMUT015812, ka mea i remarkably up-regulated i loko o kaʻiole māmā adenocarcinoma a negatively regulated e ka P53 re-expression, ua ike ia e kālailai kona cellular hana.
4. Knockdown o NONMMUT015812 e nā shRNA i hoʻemi i ka hoʻonui a me ka neʻe ʻana o nā pūnaewele KP.ʻO NONMMUT015812 kahi maʻi oncogene.
 ʻO ka nānā ʻana o ke ala KEGG o nā ʻano genes i hōʻike ʻia i loko o nā pūnaewele KP NONMMUT015812-knockdown. |  ʻO ka loiloi Gene Ontology o nā genes i hōʻike ʻokoʻa ʻia i nā pūnaewele KP NONMMUT015812-knockdown |
Kuhikuhi
Deregulated lncRNA hōʻike hōʻike i loko o ka ʻiole lung adenocarcinomas me KRAS-G12D mutation a me P53 knockout [J].Nūpepa o ka Cellular and Molecular Medicine, 2019, 23(10).DOI: 10.1111/jcmm.14584
