MRNA Fa'asologa atoa-Nanopore
Tulaga Auaunaga
● Fa'aituau fa'asologa maualalo
● Fa'aalia mole mole o le cDNA atoa
● Fa'aitiiti fa'amaumauga e mana'omia e aofia ai le numera tutusa o tusitusiga
● Fa'ailoaina o le tele o isoforms ile kene
● Fa'amatalaga fa'atusa ile tulaga isoform
Fa'amatalaga Auaunaga
| Faletusi | Fa'avae | Tuuina atu o fa'amatalaga fautuaina (Gb) | Puleaina lelei |
| cDNA-PCR(Poly-A fa'atamaoaigaina) | Nanopore PromethION P48 | 6 Gb/fa'ata'ita'iga (Fa'alagolago ile ituaiga) | Fuafuaga umi atoa＞70% Fa'ailoga tulaga lelei: Q10
|
Bioinformatics su'esu'ega
●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)
Fa'ata'ita'iga Mana'oga ma le Tu'uina atu
Fa'ata'ita'iga Mana'oga:
Nucleotides:
| Conc.(ng/μl) | Aofa'i (μg) | Mama | Amiotonu |
| ≥ 100 | ≥ 0.6 | 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 |
Tisu: Mamafa(mamago): ≥1 g
*Mo mea e la'ititi ifo i le 5 mg, matou te fautua atu e lafo atu le moli fa'aaisa (i le nitrogen vai) fa'ata'ita'iga.
Taofi fa'agata: Faitauga sela = 3×106- 1×107
* Matou te fautua atu e lafo le lysate cell frozen.I le tulaga o lena sela e laʻititi ifo nai lo le 5x105, e fautuaina le moli fa'aaisa i le nitrogen vai, lea e sili ona lelei mo le fa'apipi'iina.
Su'ega toto: Volume≥1 ml
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... ...
La'u: 2, Fa'amamago-aisa: Fa'ata'ita'iga e mana'omia ona fa'apipi'i i taga ma tanu i le aisa-mago.
- RNAstable tubes: RNA samples e mafai ona fa'amago ile RNA stabilization tube(fa'ata'ita'iga RNAstable®) ma lafo ile potu vevela.
Auala o Galuega Au'aunaga
Nucleotides:
Fa'ata'ita'iga tu'uina atu
Fauga 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
Fauga faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
1.Su'esu'ega fa'aaliga eseese -Volcano plot
O su'esu'ega fa'aaliga eseese e mafai ona fa'agasolo i tulaga uma e lua e fa'ailoa ai kenera fa'aalia eseese (DEGs) ma ile tulaga fa'aisoform e fa'ailoa ese ai.
fa'amatalaga fa'aalia (DETs)
2.Fa'afanua vevela fa'apipi'i fa'avasega
3. Alternative splicing fa'amatalaga ma fa'avasegaga
E lima ituaiga o isi mea faʻapipiʻi e mafai ona valoia e Astalavista.
4.Su'ega poly-adenylation (APA) mea e iloagofie ai ma Motif i le 50 bp i luga o le poly-A
BMK mataupu
Su'ega fa'asinomaga fa'apipi'i ma le fa'avasegaina o le isoform-level e ala ile nanopore fa'asologa fa'asologa fa'asologa atoa.
Lolomiina:Fesootaiga Natura,2020
Fuafuaga fa'asologa:
Tu'ufa'atasiga: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E suiga);3. sela B masani
Fa'asologa o fuafuaga: Fa'asologa o faletusi MiniION 2D, fa'asologa o faletusi PromethION 1D;fa'amatalaga pupuu mai fa'ata'ita'iga tutusa
Fa'asologa fa'asologa: Nanopore MinION;Nanopore PromethION;
I'uga autu
1.Isoform-level Alternative Splicing Identification
O fa'asologa umi-faitau e fa'amalosia ai le fa'ailoaina o le mutant SF3B1K700E-suia nofoaga splice i isoform-tulaga.35 isi 3'SSs ma 10 isi 5'SSs na maua e matua'i ese'ese le va o le SF3B1K700Ema le SF3B1WT.O le 33 o le 35 suiga na faatoa maua e ala ua leva ona faitau fa'asologa.
2.Isoform-tulaga Alternative Splicing quantification
Fa'aaliga o le fa'atumauina o le intron(IR) isoforms ile SF3B1K700Ema le SF3B1WTna faʻatatauina faʻavae i luga o faʻasologa o nanopore, faʻaalia ai le faʻatonutonuina o le lalolagi i lalo ole IR isoforms ile SF3B1K700E.
Fa'asinomaga
Tang AD, Soulette CM, Baren MJV, ma isi.O faʻamatalaga faʻamatalaga atoa o le suiga o le SF3B1 i le leukemia lymphocytic masani e faʻaalia ai le faʻaitiitia o le taofiofia o introns [J].Fesootaiga Natura.
