Ukulandelana kwe-mRNA okugcwele ubude
Izici
● Ukuthwebula i-poly-A mRNA kulandelwe ukuhlanganiswa kwe-cDNA kanye nokulungiswa komtapo wolwazi
● Ukulandelana kwemibhalo ephelele
● Ukuhlaziywa kwe-Bioinformatic okusekelwe ekuqondeni i-genome yokubhekisela
● Ukuhlaziywa kwe-Bioinformatic akubandakanyi nje kuphela ukuvezwa ezingeni lezakhi zofuzo kanye ne-isoform kodwa futhi nokuhlaziywa kwe-lncRNA, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation kanye nesakhiwo sezakhi zofuzo.
Izinzuzo Zesevisi
●Ukulinganiswa kokubonakaliswa ezingeni le-isoform: okuvumela ukuhlaziywa kokubonakaliswa okuningiliziwe nokunembile, okuveza ushintsho olungafihlwa lapho kuhlaziywa ukubonakaliswa kwezakhi zofuzo konke
●Izidingo Zedatha Ezincishisiwe:Uma kuqhathaniswa ne-Next-Generation Sequencing (NGS), i-Nanopore sequencing ibonisa izidingo zedatha eziphansi, okuvumela amazinga alinganayo okugcwala kwe-gene expression quantification ngedatha encane.
●Ukunemba okuphezulu kokulinganisa ukubonakaliswa: kokubili ezingeni lezakhi zofuzo kanye nele-isoform
●Ukuhlonza ulwazi olwengeziwe lwe-transcriptomic: i-polyadenylation ehlukile, izakhi zofuzo zokuhlanganiswa kanye ne-lcnRNA kanye nezakhi zofuzo zazo eziqondiwe
●Ubuchwepheshe ObubanziIthimba lethu liletha ulwazi oluningi kuyo yonke iphrojekthi, seliqede amaphrojekthi angaphezu kuka-850 e-Nanopore full-length transcriptome futhi lacubungula amasampula angaphezu kuka-8,000.
●Usekelo Lwangemva Kokuthengisa: ukuzibophezela kwethu kudlulela ngale kokuqedwa kwephrojekthi ngesikhathi sesevisi yezinyanga ezintathu ngemuva kokuthengisa. Phakathi nalesi sikhathi, sinikeza ukulandelwa kwephrojekthi, usizo lokuxazulula izinkinga, kanye nezikhathi ze-Q&A ukuze kuxazululwe noma yimiphi imibuzo ehlobene nemiphumela.
Izidingo Zesampula Nokulethwa
| Umtapo Wolwazi | Isu lokulandelana kwezigaba | Kunconywa idatha | Ikhwalithi yokulawula |
| I-Poly A ecebile | I-Nanopore PromethION 48 | 6/12 Gb | Isilinganiso sekhwalithi esimaphakathi: Q10 |
Izidingo Zesampula:
Ama-Nucleotide:
| I-Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ukungcoliswa kwephrotheni noma i-DNA okulinganiselwe noma okungekho nhlobo kukhonjisiwe kujeli. | Kwezitshalo: I-RIN≥7.0; Ezilwaneni: I-RIN≥7.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselwe noma okungekho kwesisekelo |
● Izitshalo:
Impande, Isiqu noma Ipetali: 450 mg
Iqabunga noma Imbewu: 300 mg
Izithelo: 1.2 g
● Isilwane:
Inhliziyo noma Amathumbu: 300 mg
I-Viscera noma Ubuchopho: 240 mg
Imisipha: 450 mg
Amathambo, Izinwele noma Isikhumba: 1g
● Ama-arthropod:
Izinambuzane: 6g
I-Crustacea: 300 mg
● Igazi eliphelele: Ishubhu eli-1
● Amaseli: 106 amaseli
Ukulethwa Kwesampula Okunconyiwe
Isitsha: Ishubhu le-centrifuge elingu-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)
Ukulebula kwesampula: Iqembu + ukukopisha isib. A1, A2, A3; B1, B2, B3.
Ukuthunyelwa:
1. Iqhwa elomile: Amasampula kudingeka apakishwe ezikhwameni bese egqitshwa eqhweni elomile.
2. Amashubhu e-RNAstable: Amasampula e-RNA angomiswa eshubhu lokuzinzisa i-RNA (isb. i-RNAstable®) bese ethunyelwa ekushiseni kwegumbi.
Ukugeleza Komsebenzi Wesevisi
Ama-Nucleotide:
Ukulethwa kwesampula
Ukwakhiwa komtapo wolwazi
Ukulandelana
Ukuhlaziywa kwedatha
Izinsizakalo zangemuva kokuthengisa
Ukugeleza Komsebenzi Wesevisi
Izicubu:
Umklamo wokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-RNA
Ukwakhiwa komtapo wolwazi
Ukulandelana
Ukuhlaziywa kwedatha
Izinsizakalo zangemuva kokuthengisa
● Ukucutshungulwa kwedatha eluhlaza
● Ukuhlonza umbhalo oqoshiwe
● Ukuhlanganisa okunye
● Ukulinganiswa kokubonakaliswa kwezinga lezakhi zofuzo kanye nezinga le-isoform
● Ukuhlaziywa kokubonakaliswa kokwehluka
● Isichasiselo somsebenzi kanye nokucebisa (ama-DEG nama-DET)
Ukuhlaziywa kokuhlanganisa okunye
Ukuhlaziywa Kwe-Alternative Polyadenylation (APA)
Ukubikezela kwe-lncRNA
Isichasiselo sezakhi zofuzo ezintsha
Ukuhlanganiswa kwama-DET
Amanethiwekhi Amaprotheni Namaphrotheni kuma-DEG
Hlola intuthuko eyenziwe yizinsizakalo zokulandelana kwe-mRNA ze-BMKGene ze-Nanopore ngokugcwele ngokusebenzisa iqoqo lezincwadi ezikhethiwe.
UGong, B. nabanye. (2023) 'Ukusebenza kwe-epigenetic kanye ne-transcriptional kwe-secretory kinase FAM20C njenge-oncogene ku-glioma', i-Journal of Genetics and Genomics, 50(6), amakhasi 422–433. doi: 10.1016/J.JGG.2023.01.008.
He, Z. et al. (2023) 'Ukulandelana okuphelele kwe-transcriptome yama-lymphocyte asabela ku-IFN-γ kwembula impendulo yomzimba egobekile ye-Th1 ku-flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, k. 108636. doi: 10.1016/J.FSI.2023.108636.
UMa, Y. nabanye. (2023) 'Ukuhlaziywa kokuqhathanisa kwezindlela zokulandelana kwe-PacBio kanye ne-ONT RNA zokuhlonza ubuthi be-Nemopilema Nomurai', i-Genomics, 115(6), ikhasi 110709. doi: 10.1016/J.YGENO.2023.110709.
Yu, D. et al. (2023) 'Ukuhlaziywa kwe-nano-seq kwembula ukuthambekela okuhlukile kokusebenza phakathi kwama-exosome nama-microvesicles atholakala ku-hUMSC', i-Stem Cell Research and Therapy, 14(1), amakhasi 1–13. doi: 10.1186/S13287-023-03491-5/TABLES/6.
