Fa'asologa umi e le fa'ailoga-Illumina
Tulaga Auaunaga
● Tulaga Auaunaga
● Cellular ma aano e patino
● Laasaga fa'apitoa e fa'aalia ma fa'aalia suiga fa'aalia malosi
● Fa'ata'ita'iga sa'o o taimi ma avanoa
● Suʻesuʻega faʻatasi ma faʻamaumauga mRNA.
● BMKCloud fa'avae fa'ai'uga tu'uina atu: Fa'asinomaga fa'amaumauga e maua i luga ole tulaga.
● Au'aunaga ina ua mae'a fa'atauga aoga mo le 3 masina pe a mae'a galuega
Fa'ata'ita'iga Mana'oga ma le Tu'uina atu
| Faletusi | Fa'avae | Fa'amatalaga fautuaina | Fa'amatalaga QC |
| fa'aitiitiga o le rRNA | Illumina PE150 | 10 Gb | Q30≥85% |
| Conc.(ng/μl) | Aofa'i (μg) | Mama | Amiotonu |
| ≥ 100 | ≥ 0.5 | 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≥6.5; Mo manu: RIN≥7.0; 5.0≥28S/18S≥1.0; fa'atapula'a pe leai se maualuga maualuga |
Nucleotides:
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 = 3×107
* 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.
Su'ega toto:
PA×geneBloodRNATube;
6mLTRIzol ma le 2mL toto(TRIzol:Blood=3:1)
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. Dry-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 faʻamago i le RNA stabilization tube(eg RNAstable®) ma lafo i le vevela potu.
Auala o Galuega Au'aunaga
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
Bioinformatics
1.LncRNA fa'avasegaga
LncRNA valoia e le fa softwares i luga na faavasegaina i 4 vaega: lincRNA, anti-sense-LncRNA, intronic-LncRNA;lagona-LncRNA.LncRNA fa'avasegaina na fa'aalia i le histogram i lalo.
LncRNA fa'avasegaga
2.Cis-fa'atatau i kene o DE-lncRNA su'esu'ega fa'atamaoaigaina
ClusterProfiler sa fa'afaigaluega i le GO su'esu'ega fa'atamaoaigaina i genes fa'atatau o le lncRNA (DE-lncRNA) fa'aalia eseese, e tusa ai ma faiga fa'aola, galuega mole mole ma vaega feavea'i.GO su'esu'ega fa'atamaoaigaina ose fa'agasologa e fa'ailoa ai le DEG e fa'atamaoaigaina fa'atamaoaigaina tu'utu'uga GO fa'atusatusa ile genome atoa.O faaupuga fa'atamaoaigaina na tu'uina atu ile histogram, bubble siata, ma isi e pei ona fa'aalia i lalo.
Cis-faʻatatau i kene o DE-lncRNA faʻatamaoaigaina suʻesuʻega -Bubble siata
3. I le faʻatusatusaina o le umi, numera o le exon, ORF ma le aofaʻi o faʻamatalaga o le mRNA ma le lncRNA, e mafai ona tatou malamalama i le eseesega i le fausaga, faʻasologa ma isi i le va oi latou, ma faʻamaonia foi pe o le tala lncRNA na valoia e tatou e fetaui ma uiga lautele.
BMK mataupu
Deregulated lncRNA faʻamatalaga faʻamatalaga i le isumu mama adenocarcinomas ma KRAS-G12D mutation ma P53 knockout
Lolomiina:Journal of Cellular and Molecular Medicine，2019
Fuafuaga fa'asologa
Illumina
Fa'ata'ita'iga aoina
O le NONMMUT015812-knockdown KP (shRNA-2) sela ma le pulea lelei (sh-Scr) sela na maua i le aso 6 o se faʻamaʻi viral faʻapitoa.
I'uga autu
O lenei suʻesuʻega e suʻesuʻe ai le lncRNA faʻaalia i le adenocarcinoma mama isumu ma le P53 knockout ma le KrasG12D mutation.
1.6424 lncRNAs na faʻaalia eseese (≥ 2-gaiga suiga, P <0.05).
2. I totonu o 210 lncRNAs uma (FC≥8), 11 lncRNAs 'fa'aaliga na fa'atonutonuina e P53, 33 lncRNAs e KRAS ma 13 lncRNAs e hypoxia i sela KP autu, faasologa.
3.NONMMUT015812, lea na matua'i fa'atulafonoina i le adenocarcinoma mama isumu ma le lelei fa'atonutonuina e le P53 toe fa'aalia, na iloa e fa'avasega lana galuega feavea'i.
4.Knockdown o NONMMUT015812 e shRNAs faʻaititia le faʻateleina ma le femalagaiga gafatia o sela KP.NONMMUT015812 ose oncogene ono mafai.
 KEGG ala suʻesuʻega o kenera faʻaalia eseese i totonu ole NONMMUT015812-knockdown KP sela |  Gene Ontology suʻesuʻega o kenera faʻaalia eseese i totonu ole NONMMUT015812-knockdown KP sela |
Fa'asinomaga
Deregulated lncRNA faʻamatalaga faʻamatalaga i le isumu mama adenocarcinomas ma KRAS-G12D mutation ma P53 knockout [J].Journal of Cellular and Molecular Medicine, 2019, 23(10).DOI: 10.1111/jcmm.14584
