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circRNA sequencing-Illumina

ʻO ka CircRNAs (Circular RNAs) kekahi papa o nā molekele RNA non-coding i pōkole o ka 5′ pāpale hope a me 3′ hopena poly(A) huelo.Hana nā CircRNA i ka hoʻolālā pōʻai e ka paʻa covalent kahi kūlana kūʻē i ka RNA exonuclease digestion.He kuleana koʻikoʻi ka circRNA i ka ulu ʻana a me ka hoʻomohala ʻana o nā mea ola a me ko lākou kūʻē ʻana i ke kaiapuni o waho.
He nui nā hana a CircRNA, hiki iā ia ke hoʻokūkū i ka miRNA, hoʻokō i ka hana hoʻoponopono o ceRNA, e hoʻoponopono i ka hōʻike gene.I nā makahiki i hala iho nei, ua ʻike ʻia he pili pili i ka hanana a me ka ulu ʻana o nā maʻi, a loaʻa iā ia nā manaʻo noi nui i ke kuhikuhi ʻana i nā hōʻailona hōʻailona maʻi a pēlā aku.


Nā kikoʻī lawelawe

Bioinformatics

Nā hopena hōʻike

Nānā Hiʻohiʻona

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

Laʻana QC

Hoʻolālā hoʻokolohua

hāʻawi laʻana

Hāʻawi laʻana

Hoʻokolohua hoʻokele

RNA unuhi

Hoʻomākaukau hale waihona puke

Kūkulu hale waihona puke

Hoʻomākaukau hale waihona puke

Ke kaʻina ʻana

ʻIkepili ʻikepili

ʻIkepili ʻikepili

Mahope o ke kuai ana

Nā lawelawe ma hope o ke kūʻai aku


  • Mua:
  • Aʻe:

  • Bioinformatics

    wps_doc_15

    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.

     

    Māhele-o-circRNA-host

     

    Ka mahele o ka circRNA host

    2.Clustering Analysis ma DE-circRNA
    Hierarchical-clustering-of-differentially-expressed-circRNAs

    ʻO ka hui ʻana o ka hierarchical o nā circRNA i hōʻike ʻia

    3.GO Annotation ma DE-circRNA Host Genes

    C01_vs_C02

    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

    PB-lōʻihi-RNA-Sequencing-case-study

    nā pae miRNA a me nā pae protein

    Kuhikuhi

    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

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