Imikhiqizo

Ukulandelana okuphelele kwe-transcriptome - Illumina

Ukulandelana okuphelele kwe-transcriptome - Isithombe esifakiwe se-Illumina

Ukulandelana okuphelele kwe-transcriptome kunikeza indlela ebanzi yokuphrofayilila ama-molecule e-RNA ahlukahlukene, ahlanganisa kokubili amakhodi (mRNA) nama-RNA angafaki amakhodi (lncRNA, circRNA, kanye ne-miRNA).Le nqubo ithwebula i-transcriptome ephelele yamaseli athile ngesikhathi esithile, okuvumela ukuqonda okuphelele kwezinqubo zamaselula.Okwaziwa nangokuthi "ingqikithi yokulandelana kwe-RNA," ihlose ukwambula amanethiwekhi okulawula ayinkimbinkimbi ezingeni le-transcriptome, okuvumela ukuhlaziya okujulile okufana nokuncintisana kwe-Endo native RNA (ceRNA) kanye nokuhlaziywa kwe-RNA okuhlanganyelwe.Lokhu kuphawula isinyathelo sokuqala sokwenza abalingisi abasebenzayo, ikakhulukazi ekwembuleni amanethiwekhi okulawula ahlanganisa ukusebenzisana kwe-ceRNA okusekelwe ku-circRNA-miRNA-mRNA.

Xhumana nathi

Imininingwane Yesevisi

I-Bioinformatics

Imiphumela yedemo

Okushicilelwe Okufakiwe

Izici

● Umtapo wezincwadi okabili ozolandelanisa i-transcriptome ephelele: ukuncipha kwe-rRNA okulandelwa ukulungiselelwa komtapo wezincwadi we-PE150 nokukhetha usayizi okulandelwa ukulungiselelwa komtapo we-SE50

● Qedela ukuhlaziya kwe-bioinformatics kwe-mRNA, i-lncRNA, i-circRNA ne-miRNA emibikweni ehlukene ye-bioinformatics.

● Ukuhlaziywa okuhlangene kwakho konke ukusho kwe-RNA embikweni ohlanganisiwe, okuhlanganisa nokuhlaziywa kwamanethiwekhi e-ceRNA.

Izinzuzo Zesevisi

●Ukuhlaziywa okujulile kwamanethiwekhi okulawula: Ukuhlaziywa kwenethiwekhi ye-ceRNA kunikwe amandla ukulandelana okuhlangene kwe-mRNA, i-lncRNA, i-circRNA ne-miRNA kanye nokugeleza komsebenzi okuphelele kwe-bioinformamatic.

●Isichasiselo Esiphelele: sisebenzisa imininingo egciniwe eminingi ukuze sichasise ngokusebenza Izakhi Zofuzo Ezivezwe Ngokuhlukile (ama-DEG) futhi senze ukuhlaziya okunothisayo okuhambisanayo, sinikeze imininingwane ngezinqubo zeselula nezamangqamuzana ezingaphansi kwempendulo ye-transcriptome.

●Ubungcweti Olubanzi: ngerekhodi lokuvala ngempumelelo amaphrojekthi e-transcriptome angaphezu kuka-2000 ezizindeni ezihlukene zocwaningo, ithimba lethu liletha ingcebo yesipiliyoni kuyo yonke iphrojekthi.

●Ukulawulwa Kwekhwalithi Okuqinile: sisebenzisa amaphuzu okulawula abalulekile kuzo zonke izigaba, kusukela kusampula nokulungiselela umtapo wezincwadi ukuya ekulandelaneni kanye ne-bioinformatics.Lokhu kuqapha ngokucophelela kuqinisekisa ukulethwa kwemiphumela ehlala njalo yekhwalithi ephezulu.

● Isichasiselo Esibanzi: sisebenzisa imininingo egciniwe eminingi ukuze sichasise ngokusebenza Izakhi Zofuzo Ezivezwe Ngokuhlukile (ama-DEG) futhi senze ukuhlaziya okunothisayo okuhambisanayo, sinikeze imininingwane ngezinqubo zeselula nezamangqamuzana ezingaphansi kwempendulo ye-transcriptome.

●Ukusekelwa Kwangemva Kokuthengisa: Ukuzinikela kwethu kudlulela ngalé kokuphothulwa kwephrojekthi ngenkathi yesevisi yezinyanga ezi-3 ngemva kokuthengisa.Ngalesi sikhathi, sinikezela ngokulandelelwa kwephrojekthi, usizo lokuxazulula inkinga, namaseshini e-Q&A ukuze sibhekane nanoma yimiphi imibuzo ehlobene nemiphumela.

Izidingo Zesampula Nokulethwa

Umtapowolwazi	Isu lokulandelanisa	Idatha iyanconywa	Ikhwalithi yokulawula
I-rRNA iphelile	I-Ilumina PE150	16 Gb	Q30≥85%
Usayizi ukhethiwe	I-Ilumina SE50	10-20M ukufundwa	Q30≥85%

Izimfuneko zesampula:

Ama-Nucleotides:

I-Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

≥ 100

≥ 1

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Amaprotheni anomkhawulo noma awekho nhlobo noma ukungcola kwe-DNA okuboniswa kujeli.

Izitshalo: RIN≥6.5

Isilwane: RIN≥7.0

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungenakho

Ukulethwa Kwesampula Okunconyiwe

Isitsha:
2 ml centrifuge tube (Tin foil ayinconywa)
Ukulebula okuyisampula: Ukuphindaphinda kweqembu+isb A1, A2, A3;B1, B2, B3....

Ukuthunyelwa:
1.Iqhwa elomile: Amasampula adinga ukupakishwa ezikhwameni futhi agqitshwe eqhweni elomile.
2.Amashubhu e-RNAstable: Amasampula e-RNA angomiswa ngeshubhu lokuzinzisa le-RNA(isb. i-RNAstable®) futhi athunyelwe ekamelweni lokushisa.

Ukugeleza Komsebenzi Wesevisi

Idizayini yokuhlola

Ukulethwa kwesampula

Ukukhishwa kwe-RNA

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukuhlaziywa kwedatha

Amasevisi angemuva kokuthengisa

Okwedlule: I-Chromatin Immunoprecipitation Sequencing (ChIP-seq)

Olandelayo: i-circRNA sequencing-Illumina

I-Bioinformatics

Uhlolojikelele lwesisho se-RNA

Izakhi zofuzo ezivezwe ngokwehlukana

Ukuhlaziywa kwe-ceRNA

I-miRNA evezwe ngokuhlukile nama-RNA ahlobene

Hlola intuthuko yocwaningo ekhuthazwe yi-BMKGene' yonke izinsiza zokulandelana kwe-transcriptome ngeqoqo elikhethiwe lokushicilelwe.

Dai, Y. et al.(2022) 'Amaphrofayili enkulumo abanzi we-mRNAs, lncRNAs kanye ne-miRNAs ku-Kashin-Beck isifo esihlonzwe ukulandelana kwe-RNA', i-Molecular Omics, 18(2), pp. 154–166.doi: 10.1039/D1MO00370D.

U-Liu, N. nan et al.(2022) 'Uhlaziyo oluphelele lwama-transcriptomes okumelana nokubanda kwe-Apis cerana eNtabeni yase-Changbai phakathi nesikhathi sobusika.', Gene, 830, pp. 146503–146503.doi: 10.1016/J.GENE.2022.146503.

Wang, XJ et al.(2022) 'I-Multi-Omics-Integration-Based Priorization of Competing Endogenous RNA Regulation Networks in Small Cell Lung Cancer: Characteristics of Molecular and Drug Candidates', Frontiers in Oncology, 12, p.904865. doi: 10.3389/FONC.2022.904865/BIBTEX.

Xu, P. et al.(2022) 'Ukuhlaziywa okuhlanganisiwe kwephrofayili ye-lncRNA/circRNA-miRNA-mRNA yembula imininingwane yenoveli ezindleleni ezingaba khona ekuphenduleni i-root-knot nematode kumantongomane', BMC Genomics, 23(1), pp. 1–12.doi: 10.1186/S12864-022-08470-3/AMAFIGURES/7.

Yan, Z. et al.(2022) 'Ukulandelana kwe-RNA ye-Whole-transcriptome kugqamisa izindlela zamangqamuzana ezihambisana nokugcinwa kwekhwalithi yangemva kokuvuna ku-broccoli nge-red LED irradiation', Postharvest Biology and Technology, 188, p.111878. doi: 10.1016/J.POSTHARVBIO.2022.111878.