Ogologo usoro enweghị koodu-Ilumina
Uru ọrụ
● Uru Ọrụ
● Cellular na anụ ahụ kpọmkwem
● Ogbo a kapịrị ọnụ na-egosipụta ma na-enye mgbanwe mgbanwe okwu
● Usoro nke oge na nkọwa oghere
● Nyocha njikọ na data mRNA.
● Nnyefe sitere na BMKCloud: Ngwuputa data ahaziri iche dị na ikpo okwu.
● Ọrụ ire ere gachara ọnwa 3 ka arụchara ọrụ
Ihe nlele chọrọ na nnyefe
| Ụlọ akwụkwọ | Platform | Data akwadoro | Data QC |
| mbelata rRNA | Ilumina PE150 | 10 gb | Q30≥85% |
| Conc.(ng/μl) | Ọnụ ego (μg) | Ịdị ọcha | Iguzosi ike n'ezi ihe |
| ≥ 100 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Enwere oke ma ọ bụ enweghị protein ma ọ bụ mmetọ DNA nke egosiri na gel. | Maka osisi: RIN≥6.5; Maka anụmanụ: RIN≥7.0; 5.0≥28S/18S≥1.0; nwere oke ma ọ bụ enweghị mgbago elu |
Nucleotides:
Anụ ahụ: Ibu (akọrọ): ≥1 g
* Maka anụ ahụ dị obere karịa 5 mg, anyị na-akwado iziga ihe nlele anụ ahụ kpọnwụrụ akpọnwụ (na mmiri mmiri nitrogen).
Nkwụsị cell: Ọnụ ọgụgụ cell = 3×107
* Anyị na-akwado ibupu cell lysate oyi kpọnwụrụ.Ọ bụrụ na sel ahụ riri obere karịa 5 × 105, Flash jụrụ oyi na mmiri mmiri nitrogen ka akwadoro.
Ihe nlele ọbara:
PA × geneBloodRNATube;
6mLTRIzol na 2mL ọbara (TRIzol: Blood = 3: 1)
Nnyefe nlele akwadoro
Akpa: 2 ml centrifuge tube (Tin foil adịghị akwadoro)
Ịkpọ aha nlele: Otu+ megharịa dịka A1, A2, A3;B1, B2, B3......
Mbupu:
1.Dry-ice: A ga-etinye ihe nlele n'ime akpa ma lie ya na akọrọ-ice.
2.RNAstable tubes: Enwere ike ihichapụ ihe nlele RNA na tube stabilization RNA (dịka RNAstable®) ma bufee ya na okpomọkụ.
Usoro ọrụ ọrụ
Nnwale imewe
Nbufe ihe atụ
mmịpụta RNA
Ịrụ ụlọ akwụkwọ
Usoro
Nyocha data
Mgbe-ire ọrụ
Bioinformatics
1.LncRNA nhazi ọkwa
LncRNA buru amụma site na sọftụwia anọ dị n'elu n'ụdị 4: lincRNA, anti-sense-LncRNA, intronic-LncRNA;uche-LncRNA.E gosipụtara nhazi ọkwa LncRNA na histogram dị n'okpuru.
Nhazi LncRNA
2.Cis-ezubere iche mkpụrụ ndụ ihe nketa nke DE-lncRNA enrichment analysis
Ejiri ClusterProfiler n'ọrụ na nyocha nkwalite GO na mkpụrụ ndụ ihe nketa cis ezubere iche nke lncRNA (DE-lncRNA) akọwapụtara iche, n'ihe gbasara usoro ndu, ọrụ molecular na akụrụngwa cellular.Nyocha ịba ụba GO bụ usoro iji chọpụta usoro DEG-eduzi nke ukwuu bara ụba nke GO ma e jiri ya tụnyere genome dum.E gosipụtara okwu ndị bara ụba na histogram, eserese afụ, wdg dị ka egosiri n'okpuru.
Mkpụrụ ndụ ihe nketa nke DE-lncRNA nyochara uru nke Cis-Chartị Bubble
3. Site n'ịtụle ogologo, nọmba exon, ORF na ọnụ ọgụgụ nke mRNA na lncRNA, anyị nwere ike ịghọta ọdịiche dị na nhazi, usoro na ihe ndị ọzọ n'etiti ha, ma chọpụta ma akwụkwọ akụkọ lncRNA nke anyị buru n'amụma kwekọrọ na njirimara izugbe.
Okwu BMK
Profaịlụ okwu lncRNA mebiri emebi na adenocarcinomas nku ume na mmụgharị KRAS-G12D na knockout P53
Ebipụtara:Akwụkwọ akụkọ nke Cellular and Molecular Medicine.2019
Usoro usoro
Illumina
Nchịkọta ihe atụ
Enwetara mkpụrụ ndụ NONMMUT015812-knockdown KP (shRNA-2) na sel njikwa na-adịghị mma (sh-Scr) na ụbọchị 6 nke ọrịa nje.
Nsonaazụ igodo
Ọmụmụ ihe a na-enyocha lncRNA ndị a na-egosipụta n'ụzọ na-adịghị mma na adenocarcinoma nku ume na P53 knockout na KrasG12D mutation.
E gosipụtara 1.6424 lncRNA dị iche iche (≥ mgbanwe okpukpu abụọ, P <0.05).
2.Among niile 210 lncRNAs (FC≥8), 11 lncRNA 'okwu na-achịkwa P53, 33 lncRNAs site KRAS na 13 lncRNAs site hypoxia na isi KP cell, n'otu n'otu.
3.NONMMUT015812, bụ nke a na-achịkwa nke ọma na nku ume adenocarcinoma na-adịghị mma site na P53 re-expression, achọpụtara iji nyochaa ọrụ cellular ya.
4.Knockdown nke NONMMUT015812 site na shRNA belatara mmụba na ikike mbugharị nke mkpụrụ ndụ KP.NONMMUT015812 bụ ihe nwere ike ime oncogene.
 Nyocha ụzọ KEGG nke mkpụrụ ndụ ihe nketa egosipụtara dị iche na sel NONMMUT015812-knockdown KP |  Gene Ontology nyocha nke mkpụrụ ndụ ihe nketa egosipụtara dị iche na sel NONMMUT015812-knockdown KP |
Ntụaka
Profaịlụ okwu lncRNA mebiri emebi na adenocarcinomas nku ume ya na mmụgharị KRAS-G12D na P53 knockout[J].Akwụkwọ akụkọ nke Cellular and Molecular Medicine, 2019, 23(10).DOI: 10.1111/jcmm.14584
