Tatelano ya nako e telele e sa khoutong-Illumina
Melemo ea Tšebeletso
● Melemo ea Tšebeletso
● Liselefounu le lisele tse khethehileng
● Mokhahlelo o itseng o hlahisa le ho hlahisa phetoho e matla ea polelo
● Mekhoa e nepahetseng ea nako le sebaka
● Tlhahlobo e kopanetsoeng e nang le lintlha tsa mRNA.
● Ho fana ka sephetho se thehiloeng ho BMKCloud: Morafo o hlophisitsoeng oa data o fumanehang sethaleng.
● Litšebeletso tsa ka mor'a thekiso li sebetsa likhoeli tse 3 ha morero o phethoa
Litlhoko tsa Mohlala le Phano
| Laebrari | Sethala | Lintlha tse khothalelitsoeng | Lintlha tsa QC |
| Ho fokotseha ha rRNA | Illumina PE150 | 10 Gb | Q30≥85% |
| Conc.(ng/μl) | Chelete (μg) | Bohloeki | Botšepehi |
| ≥ 100 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Tšoaetso e fokolang kapa ha e na protheine kapa DNA e bontšitsoeng ho gel. | Bakeng sa limela: RIN≥6.5; Bakeng sa liphoofolo: RIN≥7.0; 5.0≥28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
Li-nucleotides:
Sesepa: Boima ba 'mele(o omileng): ≥1 g
*Bakeng sa linama tse nyane ho feta 5 mg, re khothaletsa ho romela sampole ea lithane tse hatselitsoeng (ka naetrojene e metsi).
Ho emisoa ha lisele: Palo ea lisele = 3×107
* Re khothaletsa ho romella li-lysate tse leqhoa.Haeba sele eo e le nyane ho feta 5×105, ho kgothaletswa hore ho be le flash e hoamisitsweng ka hara metsi a naetrojene.
Mehlala ea mali:
PA×geneBloodRNATube;
6mLTRIzol le 2mL mali(TRIzol:Blood=3:1)
Tlhahiso ea Mehlala e khothalelitsoeng
Setshelo: 2 ml centrifuge tube (Tin foil ha e khothalletsoe)
Mohlala oa ho ngola: Sehlopha+katiso mohlala A1, A2, A3;B1, B2, B3....
Thomello:
1.Dry-ice: lisampole li hloka ho kenngoa ka mekotleng ebe li patoa ka leqhoa le omileng.
2.RNAstable tubes: RNA sampuli e ka omisoa ka RNA stabilization tube (mohlala, RNAstable®) le ho romelloa ka mocheso oa kamore.
Phallo ea Mosebetsi oa Ts'ebeletso
Moqapi oa liteko
Mohlala oa thomello
Tlhahiso ea RNA
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
Bioinformatics
Sehlopha sa 1.LncRNA
LncRNA e boletsoeng esale pele ke li-software tse 'nè tse ka holimo li ne li arotsoe ka lihlopha tsa 4: lincRNA, anti-sense-LncRNA, intronic-LncRNA;kutloisiso-LncRNA.Sehlopha sa LncRNA se bontšitsoe ho histogram e ka tlase.
Sehlopha sa LncRNA
2.Liphatsa tsa lefutso tse lebisitsoeng ho Cis tsa tlhahlobo ea ntlafatso ea DE-lncRNA
ClusterProfiler e hiriloe tlhahlobong ea ntlafatso ea GO mabapi le liphatsa tsa lefutso tse lebisitsoeng ho cis tsa lncRNA (DE-lncRNA), mabapi le lits'ebetso tsa baeloji, mesebetsi ea limolek'hule le likarolo tsa cellular.Tlhahlobo ea ntlafatso ea GO ke mokhoa oa ho khetholla mantsoe a GO a ntlafalitsoeng haholo a tataisoang ke DEG ha a bapisoa le genome eohle.Mantsoe a ntlafalitsoeng a hlahisitsoe ka histogram, bubble chart, joalo-joalo joalo ka ha ho bontšitsoe ka tlase.
Liphatsa tsa lefutso tse lebisitsoeng ho Cis tsa tlhahlobo ea ntlafatso ea DE-lncRNA -Bubble chart
3. Ka ho bapisa bolelele, nomoro ea exon, ORF le palo ea polelo ea mRNA le lncRNA, re ka utloisisa phapang ea sebopeho, tatellano le joalo-joalo pakeng tsa tsona, hape re netefatsa hore na buka ea lncRNA e boletsoeng esale pele ke rona e lumellana le litšobotsi tse akaretsang.
Taba ea BMK
Boemo ba polelo ea lncRNA e senyehileng ho adenocarcinomas ea mouse e nang le phetoho ea KRAS-G12D le ho kokota ha P53.
E hatisitsoe:Journal of Cellular le Molecular Medicine,2019
Leano la tatellano
Illumina
Pokello ea mohlala
Lisele tsa NONMMUT015812-knockdown KP (shRNA-2) le lisele tsa taolo e mpe (sh-Scr) li ile tsa fumanoa ka letsatsi la 6 la tšoaetso e itseng ea kokoana-hloko.
Liphetho tsa bohlokoa
Phuputso ena e batlisisa ka li-lncRNA tse hlahisoeng ka mokhoa o sa tsitsang ho mouse lung adenocarcinoma e nang le P53 knockout le KrasG12D phetoho.
Li-1.6424 lncRNA li ne li hlalosoa ka tsela e fapaneng (≥ 2-fold change, P <0.05).
2. Har'a tsohle tsa 210 lncRNAs (FC≥8), polelo ea 11 lncRNAs e ne e laoloa ke P53, 33 lncRNAs ke KRAS le 13 lncRNAs ka hypoxia liseleng tse ka sehloohong tsa KP, ka ho latellana.
3.NONMMUT015812, e neng e laoloa ka mokhoa o tsotehang ho mouse lung adenocarcinoma 'me e laoloa hampe ke P53 e hlalosang hape, e ile ea fumanoa ho hlahloba mosebetsi oa eona oa cellular.
4. Ho kokota ha NONMMUT015812 ka shRNAs ho fokolitse matla a ho ata le ho falla ha lisele tsa KP.NONMMUT015812 e ne e le oncogene e ka bang teng.
 Tlhahlobo ea tsela ea KEGG ea liphatsa tsa lefutso tse hlalositsoeng ka mokhoa o fapaneng liseleng tsa KP tsa NONMMUT015812-knockdown. |  Tlhahlobo ea Gene Ontology ea liphatsa tsa lefutso tse hlahisitsoeng ka mokhoa o fapaneng liseleng tsa KP tsa NONMMUT015812-knockdown. |
Referense
Boemo ba polelo ea lncRNA e tlositsoeng ho adenocarcinomas ea mouse e nang le phetoho ea KRAS-G12D le ho kokota P53 [J].Journal of Cellular and Molecular Medicine, 2019, 23(10).DOI: 10.1111/jcmm.14584
