Ixesha elide non-coding ulandelelwano-Illumina
Iinkonzo eziluncedo
● Izinto eziluncedo ngeNkonzo
● Iselula kunye nethishu
● Inqanaba elithile livakalisa kwaye libonise inguqu yembonakalo eguquguqukayo
● Iipateni ezichanekileyo zexesha kunye nembonakalo yendawo
● Uhlalutyo oludibeneyo kunye nedatha ye-mRNA.
● Ukuhanjiswa kweziphumo ezisekelwe kwi-BMKCloud: Ukuchithwa kwedatha ngokwezifiso ezikhoyo kwiqonga.
● Iinkonzo zasemva kokuthengiswa zisebenza iinyanga ezi-3 ekugqityweni kweprojekthi
IiMfuno zeSampulu kunye nokuhanjiswa
| Ithala leencwadi | Iqonga | Idatha ecetyiswayo | Idatha ye-QC |
| Ukuncipha kwe-rRNA | Illumina PE150 | 10 Gb | Q30≥85% |
| Conc.(ng/μl) | Imali (μg) | Ubunyulu | Ingqibelelo |
| ≥ 100 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli. | Kwizityalo: RIN≥6.5; Kwizilwanyana: RIN≥7.0; 5.0≥28S/18S≥1.0; umphakamo osisiseko okanye ongekhoyo |
IiNucleotides:
Izicubu: Ubunzima (obomileyo): ≥1 g
*Kwithishu ezincinci kuno-5 mg, sicebisa ukuba uthumele ifleshi ekhenkcezisiweyo (kwinitrogen engamanzi) isampulu yethishu.
Ukumiswa kweeseli: Ubalo lweeseli = 3×107
*Sicebisa ukuthumela i-lysate yeseli enomkhenkce.Kwimeko apho iseli ibala encinci kune-5 × 105, umbane okhenkcezisiweyo kulwelo lwenitrogen uyacetyiswa.
Iisampulu zegazi:
PA×geneBloodRNATube;
I-6mLTRIzol kunye ne-2mL yegazi(TRIzol:Igazi=3:1)
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli: 2 ml centrifuge ityhubhu (Ifoyile yeTin ayikhuthazwa)
Isampuli yokuleyibhela: Iqela+liphindaphinda umz. A1, A2, A3;B1, B2, B3... ...
Ukuthunyelwa ngenqanawa:
1.Umkhenkce owomileyo: Iisampuli kufuneka zipakishwe kwiingxowa kwaye zingcwatywe kwindawo eyomileyo.
I-2.RNAstable tubes: Iisampulu ze-RNA zingomiswa kwi-tube yokuzinzisa i-RNA (umzekelo, i-RNAstable®) kwaye ithunyelwe kwiqondo lokushisa.
Ukuhamba komsebenzi weNkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampuli
Ukukhutshwa kwe-RNA
Ukwakhiwa kwethala leencwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo emva kokuthengisa
Bioinformatics
Ukuhlelwa kwe-1.LncRNA
I-LncRNA exelwe kwangaphambili ngama-softwares amane apha ngasentla ahlelwe kwiindidi ze-4: i-lincRNA, i-anti-sense-LncRNA, i-intronic-LncRNA;ingqiqo-LncRNA.Ukuhlelwa kwe-LncRNA kuboniswe kwi-histogram engezantsi.
Ukuhlelwa kweLncRNA
I-2.Cis-targeted genes of DE-lncRNA enrichment analysis
I-ClusterProfiler yayiqeshwe kuhlalutyo lwe-GO lokutyebisa kwiijini ezijoliswe kwi-cis ze-lncRNA echazwe ngokwahlukileyo (DE-lncRNA), ngokweenkqubo zebhayoloji, imisebenzi ye-molecular kunye nezixhobo zeselula.Uhlalutyo lokutyetyiswa kwe-GO yinkqubo yokuchonga imigaqo ye-GO ephuculwe kakhulu ekhokelwa yi-DEG xa kuthelekiswa negenome epheleleyo.Amagama atyetyisiweyo aboniswe kwi-histogram, kwitshathi yeqamza, njl njl njengoko kubonisiwe ngezantsi.
I-cis-targeted genes ye-DE-lncRNA uhlalutyo lokutyebisa -Itshati yeBubble
3. Ngokuthelekisa ubude, inombolo ye-exon, i-ORF kunye nexabiso le-mRNA kunye ne-lncRNA, sinokuqonda iiyantlukwano kwisakhiwo, ulandelelwano kunye nokunye phakathi kwazo, kwaye siqinisekise nokuba inoveli ye-lncRNA eqikelelwe sithi ihambelana neempawu eziqhelekileyo.
Ityala le-BMK
Iprofayili yokubonisa ye-lncRNA engalawulekiyo kwi-adenocarcinomas yempuku ene-KRAS-G12D yokuguqulwa kunye nokunkqonkqoza kwe-P53
Ipapashiwe:Ijenali yeSelula kunye neMolekyuli yezamayeza,2019
Isicwangciso sokulandelelana
Illumina
Ingqokelela yesampuli
Iiseli ze-NONMMUT015812-knockdown KP (shRNA-2) kunye nolawulo olubi (sh-Scr) iiseli zifunyenwe ngosuku lwe-6 lwentsholongwane ethile yentsholongwane.
Iziphumo eziphambili
Olu phononongo luphanda ii-lncRNAs ezichazwe ngokungaqhelekanga kwi-adenocarcinoma yempuku yempuku ene-P53 yokunkqonkqoza kunye nokuguqulwa kwe-KrasG12D.
I-1.6424 lncRNAs yavezwa ngokwahlukileyo (≥ 2-fold change, P <0.05).
I-2.Phakathi kwayo yonke i-210 lncRNAs (FC≥8), i-11 lncRNAs 'inkcazo yayilawulwa yi-P53, i-33 lncRNAs yi-KRAS kunye ne-13 lncRNAs nge-hypoxia kwiiseli ze-KP eziphambili, ngokulandelanayo.
I-3.NONMMUT015812, eyayilawulwa ngokuphawulekayo kwi-mouse lung adenocarcinoma kwaye ilawulwa kakubi yi-P53 yokuphinda ibonakaliswe, yafunyanwa ukuba ihlalutye umsebenzi wayo weselula.
I-4.I-Knockdown ye-NONMMUT015812 nge-shRNAs yanciphisa ukwanda kunye nokukwazi ukufuduka kweeseli ze-KP.I-NONMMUT015812 yayiyinto enokubakho kwi-oncogene.
 Uhlalutyo lwendlela ye-KEGG yeendlela ezichazwe ngokwahlukileyo kwiiseli ze-KP ze-NONMMUT015812-knockdown. |  Uhlalutyo lwe-Gene Ontology yemfuza echazwe ngokwahlukileyo kwiiseli ze-KP ze-NONMMUT015812-knockdown |
Isalathiso
Iprofayile yembonakalo ye-lncRNA engalawulekiyo kwi-adenocarcinomas yempuku ene-KRAS-G12D utshintsho kunye ne-P53 yokunkqonkqoza [J].Ijenali ye-Cellular and Molecular Medicine, 2019, 23(10).I-DOI: 10.1111/jcmm.14584
