Imikhiqizo

Ukulandelana okungafaki amakhodi okude-Illumina

Ukulandelana okude okungafaki amakhodi-Isithombe Esifakiwe se-Illumina

Ama-RNA amade angafaki amakhodi (lncRNAs) awuhlobo lwama-molecule e-RNA anobude obudlula ama-200 nt, abonakala ngamandla aphansi kakhulu okubhala amakhodi.I-LncRNA, njengelungu eliyinhloko kuma-RNA angafaki amakhodi, itholakala kakhulu ku-nucleus ne-plasma.Ukuthuthukiswa kobuchwepheshe bokulandelana kanye ne-bioinformatics kwenza ukukhonjwa kwamanoveli amaningi e-lncRNA futhi kuhlobanise lawo anemisebenzi yebhayoloji.Ubufakazi obunqwabelene buphakamisa ukuthi i-lncRNA ihileleke kakhulu ekulawuleni i-epigenetic, ukulawulwa kokulotshiweyo kanye nomthethonqubo wangemva kokubhala.

Xhumana nathi

Imininingwane Yesevisi

I-Bioinformatics

Imiphumela yedemo

Ucwaningo lwesigameko

Izinzuzo Zesevisi

● Izinzuzo Zesevisi

● Iselula nethishu

● Isiteji esithile siveza futhi sethula ukuguquka kwesimo esiguqukayo

● Amaphethini anembayo esikhathi nendawo

● Ukuhlaziya okuhlangene ngedatha ye-mRNA.

● Ukulethwa kwemiphumela okususelwe ku-BMKCloud: Ukumbiwa kwedatha ngokwezifiso kutholakala endaweni yesikhulumi.

● Izinsizakalo zangemuva kokuthengisa zisebenza izinyanga ezi-3 lapho umsebenzi usuphothuliwe

Izidingo Zesampula Nokulethwa

Umtapowolwazi	Inkundla	Idatha enconyiwe	Idatha ye-QC
Ukushintsha kwe-rRNA	I-Ilumina PE150	10 Gb	Q30≥85%

Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

≥ 100

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

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

Okwezitshalo: RIN≥6.5;

Okwezilwane: RIN≥7.0;

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungenakho

Ama-Nucleotides:

Izicubu: Isisindo(somile): ≥1 g

*Ngezicubu ezincane kuno-5 mg, sincoma ukuthi uthumele isampula yezicubu eziqandisiwe (ku-nitrogen ewuketshezi).

Ukumiswa kweseli: Isibalo samaseli = 3×10⁷
*Sincoma ukuthi uthumele i-frozen cell lysate.Esimeni lapho lelo seli liba lincane kuno-5×10⁵, i-flash efriziwe ku-nitrogen ewuketshezi kuyanconywa.

Amasampula egazi:
PA×geneBloodRNATube;
I-6mLTRIzol kanye negazi elingu-2mL(TRIzol:Igazi=3:1)

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: Ukulandelana kwe-RNA encane-Illumina

Olandelayo: I-Non-Reference based mRNA Sequencing-Illumina

I-Bioinformatics

1.LncRNA ukuhlukaniswa

I-LncRNA ebikezelwe ama-softwares amane angenhla ahlukaniswe ngezigaba ze-4: i-lincRNA, i-anti-sense-LncRNA, i-intronic-LncRNA;umqondo-LncRNA.Ukuhlukaniswa kwe-LncRNA kuboniswe ku-histogram engezansi.

I-LncRNA-izigaba

Izigaba ze-LncRNA

2.Izakhi zofuzo eziqondiswe kuCis zokuhlaziywa kokunothisa kwe-DE-lncRNA

I-ClusterProfiler iqashwe ekuhlaziyeni okunothisiwe kwe-GO kuzakhi zofuzo ezihlosiwe ze-cis ze-lncRNA evezwe ngokuhlukile (DE-lncRNA), ngokwezinqubo zebhayoloji, imisebenzi yamangqamuzana nezingxenye zamaselula.Ukuhlaziywa kokucebisa kwe-GO kuyinqubo yokuhlonza amatemu e-GO ahlotshiswe ngokuphawulekayo e-DEG uma kuqhathaniswa negenome yonke.Amagama athuthukisiwe athulwe nge-histogram, ishadi lebhamuza, njll. njengoba kukhonjisiwe ngezansi.

I-Cis-target-genes-of-DE-lncRNA-enrichment-analysis--Bubble-chart Izakhi zofuzo eziqondiswe ku-Cis zokuhlaziywa kokunothisa kwe-DE-lncRNA -Ishadi lebhamuza

3. Ngokuqhathanisa ubude, inombolo ye-exon, i-ORF nenani lenkulumo ye-mRNA ne-lncRNA, singaqonda umehluko wesakhiwo, ukulandelana nokunye phakathi kwakho, futhi siqinisekise ukuthi inoveli i-lncRNA esiyibikezele ukuthi iyahambisana nezici ezivamile.

Icala le-BMK

Iphrofayili yokusho ye-lncRNA eyehlisiwe ku-adenocarcinomas yamaphaphu egundane enokuguqulwa kwe-KRAS-G12D kanye nokungqongqoza kwe-P53

Kushicilelwe:Ijenali ye-Cellular and Molecular Medicine,2019

Isu lokulandelanisa

Illumina

Iqoqo lesampula

Amaseli e-NONMMUT015812-knockdown KP (shRNA-2) namaseli okulawula okungalungile (sh-Scr) atholwe ngosuku lwesi-6 lokutheleleka ngegciwane elithile.

Imiphumela esemqoka

Lolu cwaningo luphenya i-lncRNAs evezwe ngokungafanele ku-mouse lung adenocarcinoma ene-P53 knockout kanye nokuguqulwa kwe-KrasG12D.
I-1.6424 lncRNAs yavezwa ngokuhlukile (≥ 2-fold change, P <0.05).
2.Phakathi kwawo wonke ama-lncRNA angu-210(FC≥8), inkulumo ye-11 lncRNAs ilawulwa yi-P53, 33 lncRNAs yi-KRAS kanye ne-13 lncRNAs nge-hypoxia kumaseli e-KP ayinhloko, ngokulandelanayo.
I-3.NONMMUT015812, eyayilawulwa ngokuphawulekayo ku-mouse lung adenocarcinoma futhi yalawulwa kabi ukuvezwa kabusha kwe-P53, itholwe ukuze ihlaziye umsebenzi wayo weselula.
4.Ukwehliswa kwe-NONMMUT015812 ngama-shRNA kwehlise ukusabalala namandla okufuduka kwamaseli e-KP.I-NONMMUT015812 bekuyi-oncogene engaba khona.

I-PB-full-length-RNA-Sequencing-case-study

Ukuhlaziywa kwendlela ye-KEGG yezakhi zofuzo ezivezwe ngokuhlukile kumaseli e-NONMMUT015812-knockdown KP

Ukuhlaziywa kweGene Ontology kwezakhi zofuzo ezivezwe ngokwehlukana kumaseli e-NONMMUT015812-knockdown KP

Ireferensi

Iphrofayili yokusho ye-lncRNA eyehlisiwe ku-adenocarcinomas yegundane yamaphaphu ngokushintshashintsha kwe-KRAS-G12D kanye ne-P53 knockout[J].Ijenali Yemithi Yeselula Neyengqamuzana, 2019, 23(10).I-DOI: 10.1111/jcmm.14584