Nyenyane RNA sequencing-Illumina
Melemo ea Tšebeletso
● motheo oa pele qetellong ea 5 o na le khetho e matla ea U ho fumana li-RNA tse itseng
● ponelopele ea liphatsa tsa lefutso tsa miRNA
● Lintlha li sebelisetsoa ho etsa tlhahlobo e kopanetsoeng le mRNA
● miRNA differential expression
● ponelopele ea liphatsa tsa lefutso tsa miRNA
● 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 |
| sRNA | Illumina SE50 | 10M/20M e baloa | Q30≥85% |
Litlhoko tsa Mohlala:
Li-nucleotides:
| Conc.(ng/μl) | Chelete (μg) | Bohloeki | Botšepehi |
| ≥80 | ≥ 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≥7.5; Bakeng sa liphoofolo: RIN≥8.0; 5.0≥28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
*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 ea centrifuge tube (ha e khothalletsoe ho etsa foil)
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
1.miRNA Boitsebiso
Selelekela se seng le se seng sa mokhethoa oa buka ea miRNAs e boletsoeng esale pele ke miRDeep2 e na le setšoantšo sa pdf se bonts'ang sebopeho sa eona le botebo ba tatellano.
Sebopeho sa selelekela sa miRNA le botebo ba tatellano
2.KEGG Matlafatso ea DE-miRNA Genes Targeted
Sebokeng sena, Biomarker e hlahlobile pele hore na litsela li hlahisoa ka bongata ka liphatsa tsa lefutso tse lebisitsoeng ho DE-miRNA.Lintlha tsa ntlafatso le tlhahlobo ea batšoasi ba litlhapi li ile tsa sebelisoa ho fumana tekanyo ea ho ntlafatsa le bohlokoa ba tsela.Equation ea lipalo tsa ntlafatso e bontšitsoe ka tlase.
Ntlafatso ea tsela ea KEGG ho liphatsa tsa lefutso tse lebisitsoeng ho DE-miRNA
Tlhahlobo ea 3.GO ea Matlafatso ho DE-miRNA Genes Targeted
clusterProfiler e hiriloe tlhahlobong ea ntlafatso ea GO ho liphatsa tsa lefutso tse lebisitsoeng ho DE-miRNA.Kerafo e lebisitsoeng ea acyclic ea mantsoe a ntlafalitsoeng e ile ea hlahisoa ho bonts'a sebopeho sa maemo a phahameng a mantsoe.Setšoantšong, tataiso ea metsu e emela likamano tsa kenyelletso pakeng tsa mantsoe, ke hore, li-node li totobetse ho feta li-node tsa tsona tse ka holimo.Kerafo ea acylic e lebisitsoeng ea liphatsa tsa lefutso tse lebisitsoeng ho DE-miRNA e bontšitsoe setšoantšong se ka tlase.
TopGO e lebisitse acyclic graph ea DE-miRNA liphatsa tsa lefutso tse lebisitsoeng
Taba ea BMK
Tlhahlobo e Kopanetsoeng ea MiRNA le Liphatsa tsa lefutso tse Amahanngoang le Boleng ba Nama e senola hore Gga-MiR-140-5p e Ama Mafura a Intramuscular ka Likhoho.
E hatisitsoe:Cellular Physiology le Biochemistry,2018
Liphetho tsa bohlokoa
Likhoho tsa morao-rao tsa nako ea ho behela li bonts'itse maemo a tlase lefatšeng ka bophara a miRNAs ho feta likhoho tse nyane.
Khokahano ea taolo ea mirNA-mrna e ka amang boleng ba nama e ile ea etsoa
gga-miR-140-5p e khothalletsa phapang ea adipocyte ka ho theola RXRG.
 gga-miR-140-5p e khothaletsa phapang ea likhoho tsa instramuscular preadipocyte ka ho shebana le 3'UTR ea RXRG |
Referense
Zhang M , Li DH , Li F , et al.Tlhahlobo e Kopanetsoeng ea MiRNA le Liphatsa tsa lefutso tse Amanang le Boleng ba Nama li Senola hore Gga-MiR-140-5p e Ama Ho Ingoa ha Mafura a Intramuscular ka Likhoho[J].Cellular Physiology le Biochemistry, 2018:2421-2433.DOI: 10.1159/000489649
