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Zogulitsa

Kutsata kwa CircRNA-Illumina

Chithunzi Chodziwika cha CircRNA Sequencing-Illumina

Kusanthula kwa RNA yozungulira (circRNA-seq) kumalemba ndikuwunika ma RNA ozungulira, gulu la mamolekyu a RNA omwe amapanga ma loops otsekedwa chifukwa cha zochitika zosavomerezeka, zomwe zimapangitsa kuti RNA iyi ikhale yolimba kwambiri. Ngakhale ma circRNA ena awonetsedwa kuti amagwira ntchito ngati masiponji a microRNA, akumagawa ma microRNA ndikuletsa kuti asayang'anire ma mRNA awo omwe akufuna, ma circRNA ena amatha kuyanjana ndi mapuloteni, kusintha mawonekedwe a majini, kapena kukhala ndi maudindo mu njira zamaselo. Kusanthula kwa mawonekedwe a circRNA kumapereka chidziwitso pa ntchito zowongolera za mamolekyu awa ndi kufunika kwawo m'njira zosiyanasiyana zamaselo, magawo okukula, ndi matenda, zomwe zimathandiza kumvetsetsa bwino zovuta za malamulo a RNA pankhani yokhudza mawonekedwe a majini.

Pulatifomu: Illumina Novaseq X

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Bioinformatics

Zotsatira za Chiwonetsero

Mabuku Odziwika

Mawonekedwe

● Kuchepa kwa rRNA kutsatiridwa ndi kukonzekera laibulale yolunjika, zomwe zimathandiza kuti deta yotsatizana ndi chingwe ipezeke.

● Njira yogwirira ntchito ya Bioinformatic imalola kulosera kwa circRNA ndi kuwerengera mawu.

Ubwino wa Utumiki

●Ukatswiri WochulukaTagwiritsa ntchito zitsanzo zoposa 20,000, zomwe zikuphatikiza mitundu yosiyanasiyana ya zitsanzo, ndipo tabweretsa ukadaulo wochuluka ku polojekiti iliyonse.

●Kulamulira Kwabwino Kwambiri: Timagwiritsa ntchito mfundo zoyendetsera zinthu m'magawo onse, kuyambira kukonzekera zitsanzo mpaka kukonzekera laibulale, kutsata malamulo ndi bioinformatics. Kuyang'anira kwathu mosamala kumatsimikizira kuti zotsatira zabwino nthawi zonse zimaperekedwa.

●Malaibulale Okwanira a RNA:Timagwiritsa ntchito rRNA depletion m'malo mwa linear RNA depletion mu kukonzekera kwathu koyambirira kwa laibulale, kuonetsetsa kuti deta yotsatizana ikuphatikizapo osati circRNA yokha komanso mRNA ndi lncRNA, zomwe zimathandiza kusanthula pamodzi kwa deta iyi.

●Kusanthula Kosankha kwa Ma Network Opikisana a Endogenous RNA (ceRNA): Kupereka chidziwitso chakuya pa njira zowongolera maselo.

● Thandizo Pambuyo pa Kugulitsa: Timamvetsetsa kufunika kokhalapo, ndichifukwa chake kudzipereka kwathu sikupitirira kutha kwa polojekiti ndi nthawi yautumiki wa miyezi itatu pambuyo pogulitsa. Panthawiyi, timapereka chithandizo chotsata polojekiti, thandizo lothetsera mavuto, ndi magawo a mafunso ndi mayankho kuti tiyankhe mafunso aliwonse okhudzana ndi zotsatira zake.

Zofunikira pa Zitsanzo ndi Kutumiza

Laibulale	Nsanja	Deta yovomerezeka	Deta QC
laibulale yowongolera ya rRNA yochepetsedwa	Illumina PE150	16-20 Gb	Q30≥85%

Zofunikira pa Zitsanzo:

Nyukleotidi:

Conc.(ng/μl)

Kuchuluka (μg)

Chiyero

Umphumphu

≥ 80

≥ 0.8

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Kuipitsidwa kwa mapuloteni kapena DNA komwe kumaoneka pang'ono kapena kosaoneka pa gel.

RIN≥6.0;

5.0≥28S/18S≥1.0;

kukwera koyambira kochepa kapena kopanda

● Zomera:

Muzu, Tsinde kapena Petal: 450 mg

Tsamba kapena Mbewu: 300 mg

Chipatso: 1.2 g

● Nyama:

Mtima kapena Matumbo: 450 mg

Viscera kapena Ubongo: 240 mg

Minofu: 600 mg

Mafupa, Tsitsi kapena Khungu: 1.5g

● Ma Arthropod:

Tizilombo: 9g

Crustacea: 450 mg

● Magazi athunthu:Machubu awiri

● Maselo: 10⁶maselo

● Seramu ndi Plasma: 6 ml

Kutumiza Zitsanzo Koyenera

Chidebe: 2 ml chubu cha centrifuge (Sikoyenera kugwiritsa ntchito pepala la zitini)

Zitsanzo zolembera: Gulu + koperani mwachitsanzo A1, A2, A3; B1, B2, B3.

Kutumiza:

1. Ayezi wouma: Zitsanzo ziyenera kuyikidwa m'matumba ndikuyikidwa mu ayezi wouma.

2. Machubu okhazikika a RNA: Zitsanzo za RNA zitha kuumitsidwa mu chubu chokhazikika cha RNA (monga RNAstable®) ndikutumizidwa kutentha kwa chipinda.

Kayendedwe ka Ntchito Yotumikira

Kapangidwe ka kuyesera

Kutumiza zitsanzo

Kuchotsa RNA

Kumanga laibulale

Kutsatana

Kusanthula deta

Ntchito zogulitsa pambuyo pogulitsa

Yapitayi: Kutsatana Kwathunthu kwa Transcriptome - Illumina

Ena: Kusanthula kwa RNA Yaing'ono-Illumina

Bioinformatics

Deta QC
Kulinganiza majini
Kuzindikira kwa CircRNA
Kufotokozera kwa CircRNA
Kusanthula kwa kusiyana kwa mawu

Kuneneratu kwa circRNA: kugawa kwa chromosome

Ma circRNA Ofotokozedwa Mosiyanasiyana - Chiwembu cha Volcano

Ma circRNA ofotokozedwa mosiyana - magulu ozungulira

Kuchulukitsa kwa majini a circRNA omwe amagwira ntchito

Fufuzani kupita patsogolo kwa kafukufuku komwe kwathandizidwa ndi ntchito za BMKGene' circRNA sequencing kudzera mu zofalitsa zosiyanasiyana.

Wang, X. et al. (2021) 'CPSF4 imalamulira kupangika kwa circRNA ndi kuletsa majini omwe amayendetsedwa ndi microRNA mu khansa ya chiwindi',Oncogene2021 40:25, 40(25), pp. 4338–4351. doi: 10.1038/s41388-021-01867-6.

Xia, K. et al. (2023) 'X oo-responsive transcriptome ikuwonetsa ntchito ya RNA133 yozungulira pakukana matenda mwa kuwongolera kuwonetsedwa kwa OsARAB mu mpunga',Kafukufuku wa Zamoyo, 5(1), tsamba 1–14. doi: 10.1186/S42483-023-00188-8/FIGURES/6.

Y, H. et al. (2023) 'CPSF3 imasintha momwe zizindikiro zozungulira ndi zolunjika zimakhalira mu khansa ya chiwindi'. doi: 10.21203/RS.3.RS-2418311/V1.

Zhang, Y. et al. (2023) 'Kuwunika kwathunthu kwa ma circRNA mu matenda a mtima a cirrhotic asanayambe komanso atapatsidwa chiwindi',Mankhwala Oteteza Matenda Padziko Lonse, 114 p. 109495. doi: 10.1016/J.INTIMP.2022.109495.