Imikhiqizo

Ukulandelana kwe-RNA Encane ye-Exosomal-Illumina

Isithombe Esifakiwe se-Exosomal Small RNA Sequencing-Illumina

Ama-Exosomes angama-vesicles amancane akhishwa amaseli, ngokuvamile asukela kuma-nanometers angu-30 kuya kwangu-100. Lawa ma-vesicles aqukethe i-RNA ehlukahlukene. Phakathi kwezinhlobo ze-RNA kuma-exosomes, evame kakhulu futhi efundwa kabanzi yi-microRNA (i-miRNA). I-miRNA iyiklasi yama-RNA amancane angabhalisi amakhodi angaba ngu-18-25 nucleotides ubude. Aqondisa ukuthuliswa kwezakhi zofuzo ngemva kokubhalwa ngokubopha esifundeni esingahunyushwanga esingu-3′ (3′ UTR) sama-mRNA aqondiwe, ngaleyo ndlela elawula ukuvezwa kwezakhi zofuzo. Isibonelo, ama-exosomes akhishwa amaseli athile esimila aqukethe ama-miRNA athile, njenge-miR-126 kanye ne-miR-92a. Lawa ma-miRNA angathonya ukuvezwa kwezakhi zofuzo kumaseli amukelayo futhi akhuthaze i-angiogenesis yesisu (uTomohiro Umezu, et al., Oncogene, 2012).

Xhumana nathi

Imininingwane Yesevisi

i-bioinformatics

Imiphumela Yedemo

Izici

● I-RNA iyonke ye-Exosomal isetshenziswa njengezinto zokufaka. Ngesikhathi sokuhlanzwa, ama-miRNA Beads asetshenziswa ukuhlukanisa i-miRNA.

● Ukuhlaziywa kwe-Bioinformatic kugxile ekubikezelweni kwe-miRNA kanye nezinhloso zayo.

Izinzuzo Zesevisi

● Ukuhlaziywa okuphelele kwe-bioinformatics: Ukuvumela ukuhlonza ama-miRNA aziwayo kanye namasha, ukuhlonza izinhloso zama-miRNA, kanye nokuchazwa okusebenzayo okuhambisanayo kanye nokucebisa ngezizindalwazi eziningi (KEGG, GO).

● Ukulawulwa Kwekhwalithi Okuqinile: Sisebenzisa amaphuzu okulawula ayisisekelo kuzo zonke izigaba, kusukela ekulungiseleleni amasampula kanye nomtapo wolwazi kuya ekulandeleni nasekubhaleni ulwazi nge-bioinformatics. Lokhu kuqapha ngokucophelela kuqinisekisa ukulethwa kwemiphumela esezingeni eliphezulu njalo.

● Ukusekelwa Ngemva Kokuthengisa: Ukuzibophezela kwethu kudlulela ngale kokuqedwa kwephrojekthi ngesikhathi sesevisi yezinyanga ezintathu ngemuva kokuthengisa. Phakathi nalesi sikhathi, sinikeza ukulandelwa kwephrojekthi, usizo lokuxazulula izinkinga, kanye nezikhathi zemibuzo nezimpendulo ukuze kuxazululwe noma yimiphi imibuzo ehlobene nemiphumela.

Izidingo Zesampula Nokulethwa

Umtapo Wolwazi	Ipulatifomu	Idatha enconyiwe	Idatha ye-QC
Ukuhlukaniswa kwe-miRNA ye-Exosomal	I-Illumina SE50	Ukufundwa okungu-10M-20M	Q30≥85%

Izidingo Zesampula

Ama-Nucleotide:

Inani (ng)	Ivolumu (μL)
40	20

Izicubu:

I-Exosome: 100 μL; 40 μg; >10^9 izinhlayiya/mL
I-Serum/plasma: 5 mL
Isithako esingaphezu kwesijwayelekile se-cell culture: 100 mL
Umchamo: 100 mL
Amathe: 15 mL
Ukuphuma kwempumulo: 15 mL
Uketshezi lwe-cerebrospinal: 15 mL
Uketshezi lwe-Amniotic: 50 mL
Isidoda: 5 mL
Inyongo: 5 mL
Ama-Ascites: 50 mL
Ubisi: 15 mL
Uketshezi lwe-Follicular: 15 mL
I-supernatant yendle: 50 mL
Isithako esikhulu se-microbial culture: 100 mL
Izicubu zezilwane: 3 g
Izicubu zesitshalo: 1 g

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

Umklamo wokuhlola

Ukulethwa kwesampula

Ukukhishwa kwe-RNA

Ukwakhiwa komtapo wolwazi

Ukulandelana

Ukuhlaziywa kwedatha

Izinsizakalo zangemuva kokuthengisa

Okwedlule: I-BMKMANU S3000_Spatial Transcriptome

Olandelayo: I-Exosomal mRNA/LncRNA/CirrcRNA Sequencing-Illumina

I-Bioinformatics

● Ukulawulwa kwekhwalithi yedatha eluhlaza
● ukuhlukaniswa kwe-sRNA
● Ukuqondanisa ne-genome yokubhekisela
● Ukuhlonza i-miRNA eyaziwayo nentsha
● Ukuhlaziywa kokubonakaliswa kwe-miRNA okuhlukile
● Isichasiselo esisebenzayo sama-targets e-miRNA

Ukuhlonza i-miRNA: isakhiwo kanye nokujula