Ka Hoʻonohonoho ʻana o ka mRNA piha-Nanopore
Nā hiʻohiʻona
● Hopu ʻia o ka poly-A mRNA a ukali ʻia e ka synthesis cDNA a me ka hoʻomākaukau ʻana o ka waihona puke
● Ka hoʻonohonoho ʻana o nā palapala piha piha
● Ka loiloi ʻikepili bioinformatic e pili ana i ka hoʻohālikelike ʻana i kahi genome kuhikuhi
● ʻAʻole wale ka loiloi Bioinformatic e pili ana i ka hōʻike ʻana ma ka pae gene a me ka isoform akā ʻo ka loiloi pū kekahi o ka lncRNA, nā fusions gene, poly-adenylation a me ke ʻano o ka gene
Nā Pōmaikaʻi o ka Lawelawe
●Ka helu ʻana o ka hōʻike ʻana ma ka pae isoform: e hiki ai ke nānā pono a pololei i ka hōʻike ʻana, e hōʻike ana i ka loli i hiki ke uhi ʻia i ka wā e nānā ai i ka hōʻike ʻana o ka gene holoʻokoʻa
●Hoʻemi ʻia nā koi ʻikepili:Ke hoʻohālikelike ʻia me ka Next-Generation Sequencing (NGS), hōʻike ka Nanopore sequencing i nā koi ʻikepili haʻahaʻa, e ʻae ana i nā pae like o ka saturation quantification expression gene me ka ʻikepili liʻiliʻi.
●ʻOi aku ka pololei o ka helu ʻana o ka hōʻike ʻana: ma ka pae gene a me ka isoform
●Ka ʻike ʻana i ka ʻike transcriptomic hou aʻe: polyadenylation ʻē aʻe, nā genes fusion a me lcnRNA a me kā lākou mau genes i manaʻo ʻia
●Ka ʻIke LoaLawe mai kā mākou hui i ka nui o ka ʻike i kēlā me kēia papahana, ua hoʻopau ma mua o 850 mau papahana transcriptome piha o Nanopore a ua hana ma mua o 8,000 mau laʻana.
●Kākoʻo Ma hope o ke kūʻai akuʻOi aku kā mākou kūpaʻa ma mua o ka hoʻopau ʻana o ka papahana me kahi manawa lawelawe ma hope o ke kūʻai aku he 3 mahina. I loko o kēia manawa, hāʻawi mākou i ka hahai ʻana i ka papahana, ke kōkua hoʻoponopono pilikia, a me nā hālāwai Q&A e pane i nā nīnau e pili ana i nā hopena.
Nā Pono Laʻana a me ka Hoʻouna ʻana
| Hale Waihona Puke | Hoʻolālā hoʻonohonoho | ʻIkepili i ʻōlelo ʻia | Ka Mana Kūlana |
| Hoʻonui ʻia ʻo Poly A | ʻO Nanopore PromethION 48 | 6/12 Gb | Ka helu maikaʻi awelika: Q10 |
Nā Pono Laʻana:
Nā Nukleotida:
| ʻO ka huina (ng/μl) | Ka nui (μg) | Maʻemaʻe | Kūpaʻa |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ua kaupalena ʻia a ʻaʻohe paha haumia protein a DNA i hōʻike ʻia ma ka gel. | No nā mea kanu: RIN≥7.0; No nā holoholona: RIN≥7.5; 5.0≥28S/18S≥1.0; palena a ʻaʻohe kiʻekiʻe kumu |
● Nā mea kanu:
Aʻa, Kumu a i ʻole Petal: 450 mg
Lau a hua paha: 300 mg
Hua: 1.2 g
● Holoholona:
ʻŌpū a i ʻole ka ʻōpū: 300 mg
ʻO loko o ke kino a i ʻole ka lolo: 240 mg
ʻIʻo: 450 mg
Iwi, lauoho a ʻili paha: 1g
● Nā ʻArthropod:
Nā pepeke: 6g
ʻŌpū: 300 mg
● Koko holoʻokoʻa1 paipu
● Nā pūnaewele106 nā pūnaewele
Hoʻouna Laʻana i Manaʻo ʻia
Pahu: ʻŌmole centrifuge 2 ml (ʻAʻole ʻōlelo ʻia ka pepa kini)
Lepili laʻana: Hui+e hana hou e laʻa A1, A2, A3; B1, B2, B3.
Hoʻouna ʻia:
1. Hau maloʻo: Pono e hoʻopaʻa ʻia nā hāpana i loko o nā ʻeke a kanu ʻia i loko o ka hau maloʻo.
2. Nā paipu RNAstable: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA i loko o ka paipu hoʻopaʻa RNA (e laʻa me RNAstable®) a hoʻouna ʻia i ka mahana o ka lumi.
Kahe Hana Lawelawe
Nā Nukleotida:
Hoʻouna ʻana i nā laʻana
Ke kūkulu ʻana i ka hale waihona puke
Ka hoʻonohonoho ʻana
Ka loiloi ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
Kahe Hana Lawelawe
ʻIʻo:
Hoʻolālā hoʻokolohua
Hoʻouna ʻana i nā laʻana
Ka unuhi ʻana o ka RNA
Ke kūkulu ʻana i ka hale waihona puke
Ka hoʻonohonoho ʻana
Ka loiloi ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
● Ka hana ʻana i ka ʻikepili maka
● Ka ʻike ʻana i ka palapala hoʻoili
● Hoʻopili ʻē aʻe
● Ka helu ʻana o ka hōʻike ʻana ma ka pae gene a me ka pae isoform
● Ka loiloi ʻana i ka hōʻike ʻokoʻa
● Ka hōʻike ʻana a me ka hoʻonui ʻana i ka hana (nā DEG a me nā DET)
Ka loiloi splicing ʻē aʻe
Ka Nānā ʻana i ka Polyadenylation ʻē aʻe (APA)
Wānana lncRNA
Ka wehewehe ʻana o nā genes hou
Ka hui ʻana o nā DET
Nā Pūnaewele Protein-Protein ma nā DEG
E ʻimi i nā holomua i kōkua ʻia e nā lawelawe hoʻonohonoho mRNA piha ʻo Nanopore a BMKGene ma o kahi hōʻiliʻili o nā puke i hoʻonohonoho pono ʻia.
ʻO Gong, B. et al. (2023) 'Hoʻoulu ʻana o ka epigenetic a me ka transcriptional o ka kinase secretory FAM20C ma ke ʻano he oncogene i loko o glioma', Journal of Genetics and Genomics, 50(6), pp. 422–433. doi: 10.1016/J.JGG.2023.01.008.
ʻO ia, Z. et al. (2023) 'ʻO ka hoʻonohonoho ʻana o ka transcriptome piha o nā lymphocytes e pane iā IFN-γ e hōʻike ana i kahi pane pale ʻole Th1-skewed i loko o ka flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.
Ma, Y. et al. (2023) 'Ka loiloi hoʻohālikelike o nā ʻano hana hoʻonohonoho ʻana o PacBio a me ONT RNA no ka ʻike ʻana i ka venom Nemopilema Nomurai', Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.
ʻO Yu, D. et al. (2023) 'Hōʻike ka loiloi Nano-seq i nā ʻano hana like ʻole ma waena o nā exosomes a me nā microvesicles i loaʻa mai hUMSC', Stem Cell Research and Therapy, 14(1), pp. 1–13. doi: 10.1186/S13287-023-03491-5/TABLES/6.
