MRNA Fa'asologa atoa-Nanopore
Vaega
● Pu'eina o le poly-A mRNA soso'o ai ma le cDNA synthesis ma tapenaga faletusi
● Fa'asologa o fa'amaumauga atoa
● Su'esu'ega o le bioinformatic e fa'atatau i le fa'aogaina o se genome fa'asino
● Bioinformatic su'esu'ega e aofia ai e le gata o fa'amatalaga ile gene ma le isoform-level ae fa'apea fo'i le su'esu'eina o lncRNA, fa'atupu fusuaga, poly-adenylation ma le fa'atulagaina o gene.
Tulaga Auaunaga
●Fuafuaga o fa'amatalaga ile tulaga isoform: mafai ai ona au'ili'ili fa'amatalaga au'ili'ili ma sa'o, fa'aalia suiga e ono fa'amalu pe a su'esu'eina le fa'aaliga atoa o kene.
●Fa'aitiitia Mana'oga o Fa'amatalaga:Pe a faatusatusa i le Next-Generation Sequencing (NGS), o le Nanopore sequencing o loʻo faʻaalia ai le maualalo o manaʻoga o faʻamaumauga, faʻatagaina mo tulaga tutusa o faʻamatalaga faʻasologa o faʻamatalaga faʻatusatusaina ma faʻamatalaga laiti.
●Sili atu le sa'o o fa'amatalaga fa'atusatusaga: i le gene ma le isoform level
●Fa'ailoaina o fa'amatalaga transcriptomic fa'aopoopo: isi polyadenylation, fusion genes ma lcnRNA ma a latou kenera taulaʻi
●Tomai Tele: O la matou 'au e aumaia le tele o le poto masani i galuega uma, ina ua maeʻa le sili atu i le 850 Nanopore faʻataʻitaʻiga faʻasologa atoa ma faʻatautaia le 8,000 faʻataʻitaʻiga.
●Lagolago Post-Fa'atau: o la matou tautinoga e faʻalautele atu i tua atu o le faʻamaeʻaina o galuega faʻatasi ma le 3-masina pe a maeʻa le faʻatau atu taimi auaunaga. I le taimi lea, matou te ofoina atu le tulitatao o poloketi, fesoasoani faʻafitauli, ma Q&A sauniga e faʻafeiloaʻi ai soʻo se fesili e fesoʻotaʻi ma iʻuga.
Fa'ata'ita'iga Mana'oga ma le Tu'uina atu
| Faletusi | Fuafuaga fa'asologa | Fa'amatalaga fautuaina | Puleaina lelei |
| Poly A fa'atamaoaigaina | Illumina PE150 | 6/12 Gb | Fa'ailoga tulaga lelei: Q10 |
Fa'ata'ita'iga Mana'oga:
Nucleotides:
| Conc.(ng/μl) | Aofa'i (μg) | Mama | Fa'amaoni |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Fa'atapula'a pe leai se polotini po'o le fa'aleagaina o DNA o lo'o fa'aalia ile gel. | Mo laau toto: RIN≥7.0; Mo manu: RIN≥7.5; 5.0≥28S/18S≥1.0; fa'atapula'a pe leai se maualuga maualuga |
● La'au:
A'a, Po'o Po'o Petal: 450 mg
Laulaau po'o Fatu: 300 mg
Fualaau: 1.2 g
● Manu:
FOTO po'o le Tonu: 300 mg
Viscera po'o Fai'ai: 240 mg
Muso: 450 mg
Ivi, Laulu po'o Pa'u: 1g
● Arthropods:
Iniseti: 6g
Crustacea: 300 mg
● toto atoa: 1 paipa
● Selula: 106 sela
Fautuaina Fa'ata'ita'iga Tu'uina atu
Containe: 2 ml centrifuge tube (E le fautuaina le pepa apa)
Fa'ailoga fa'ata'ita'iga: Vaega+fa'atusa eg A1, A2, A3; B1, B2, B3.
utaina:
1. Fa'amamago-aisa: Fa'ata'ita'iga e mana'omia ona fa'apipi'i i taga ma tanu i le aisa-mago.
2. RNAstable tubes: RNA samples e mafai ona fa'amago ile RNA stabilization tube(eg RNAstable®) ma lafo ile potu vevela.
Auala o Galuega Au'aunaga
Nucleotides:
Fa'ata'ita'iga tu'uina atu
Fausia faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
Auala o Galuega Au'aunaga
Tisu:
Fa'ata'ita'iga mamanu
Fa'ata'ita'iga tu'uina atu
RNA su'ega
Fausia faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
● Fa'asologa o fa'amatalaga mata'utia
● Faʻamatalaga faʻamatalaga
● Su'ega fa'aopoopo
● Fa'amatalaga fa'atusa i le tulaga o le kenera ma le maualuga o le isoform
● Su'esu'ega fa'aaliga eseese
● Fa'amatalaga galuega ma fa'atamaoaigaina (DEGs ma DETs)
Su'esu'ega tu'ufa'atasiga
Alternative Polyadenylation Analysis (APA)
lncRNA vavalo
Fa'amatalaga o kenera fou
Fa'avasegaina o DETs
Polotini-Protein Networks i DEGs
Su'esu'e le aga'i i luma na fa'afaigofieina e le BMKGene's Nanopore atoa le mRNA fa'asologa o auaunaga e ala i le aoina o fa'asalalauga.
Gong, B. et al. (2023) 'Epigenetic and transcriptional activation of the secretory kinase FAM20C as a oncogene in glioma', Journal of Genetics and Genomics, 50 (6), itulau 422-433. doi: 10.1016/J.JGG.2023.01.008.
Ia, Z. et al. (2023) 'Full-length transcriptome sequencing of lymphocytes tali atu i le IFN-γ faʻaalia se Th1-skewed immune response in flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.
Ma, Y. et al. (2023) 'Su'esu'ega fa'atusatusa o PacBio ma ONT RNA fa'asologa o metotia mo le fa'ailoaina o mea oona a Nemopilema Nomurai', Genomics, 115(6), i. 110709. doi: 10.1016/J.YGENO.2023.110709.
Yu, D. et al. (2023) 'Nano-seq su'esu'ega e fa'aalia ai uiga eseese o galuega i le va o exosomes ma microvesicles e maua mai i le hUMSC', Stem Cell Research and Therapy, 14(1), itulau 1–13. doi: 10.1186/S13287-023-03491-5/LAFONO/6.
