circRNA sequencing-Illumina
Pono lawelawe
● Hoʻopili nui ʻia ʻo CircRNA me ka hoʻoponopono hōʻike mRNA
● Ua komo i loko o ka ʻōnaehana hoʻoponopono o circRNA - miRNA - mRNA
● Identification and statistics on known circRNA and novel circRNA prediction
● 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 | 16 Gb | Q30≥85% |
Nucleotides:
| 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 |
Nā Koina Laʻana:
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:Koko=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:
ʻO ka hau maloʻo: Pono e hoʻopili ʻia nā laʻana i loko o nā ʻeke a kanu ʻia i loko o ka hau maloʻo.
Nā paipu RNAstable: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA i ka paipu hoʻopaʻa RNA (e laʻa me RNAstable®) a hoʻouna ʻia i loko o ka wela lumi.
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.CircRNA Hosts
Ua hōʻike ʻia ʻo ka hapa nui o nā circRNA mai nā exons ma mua o nā introns a i ʻole nā wahi intergenic.ʻOi aku ka nui o ka hāʻawi ʻana i ka circRNA ma waena o nā chromosomes.No laila, he mea waiwai nui ka loiloi ʻana i nā puʻunaue like ʻole o nā pūʻali circRNA ma waena o nā laʻana like ʻole, me ka hāʻawi ʻana i kēlā ma nā ʻāpana gene a me nā chromosomes like ʻole.
Ka mahele o ka circRNA host
2.Clustering Analysis ma DE-circRNA
ʻO ka hui ʻana o ka hierarchical o nā circRNA i hōʻike ʻia
3.GO Annotation ma DE-circRNA Host Genes
Hoʻokaʻawale GO o nā genes host DE-circRNA
Hihia BMK
Hoʻoponopono ʻo CPSF4 i ka hoʻokumu ʻana o ka circRNA a me ka microRNA mediated gene silencing i ka maʻi hepatocellular carcinoma
Paʻi ʻia: Oncogene,2021
Nā hopena koʻikoʻi
Hoʻoponopono ʻia ka ʻōlelo 1.CPSF4 i ka HCC a ua pili kēlā ʻōlelo kiʻekiʻe o CPSF4 me ka prognosis maikaʻi ʻole i nā maʻi HCC
2. Hoʻemi ka CPSF4 i nā pae o nā circRNAs, nona kahi hōʻailona hōʻailona polyadenylation a ʻo kēia emi ʻana o nā circRNA e hōʻemi ana i ka hōʻiliʻili ʻana o ka miRNA a hoʻopau i ka miRNA-mediated gene silencing ma HCC
3. Hōʻike nā hoʻokolohua i ka moʻomeheu cell a me nā kiʻi kiʻi xenograft i ka CPSF4 e hoʻolaha i ka hoʻonui ʻana o nā cell HCC a hoʻonui i ka tumorigenicity.Eia kekahi, ke kū'ē nei ka CPSF4 i ka hopena suppressor tumor o kona circRNA i lalo i ka HCC
 nā pae miRNA a me nā pae protein |
Wang X , Dong J , Li X , et al.Hoʻoponopono ʻo CPSF4 i ka hoʻokumu ʻana i ka circRNA a me ka microRNA mediated gene silencing i ka maʻi hepatocellular carcinoma [J].Oncogene.DOI: 10.1038/s41388-021-01867-6
