ABSTRACT: Analysis of expression profile of human GEN2.2 cell line RNA expression profile of the human pDC cell line GEN2.2 Total RNA was isolated from GEN2.2 cells growing at log phase with High Pure RNA isolation Kit (Roche Diagnostics, Mannheim, Germany), according with the manufacturer´s instructions in quintuplicate. A total of 300 ng of total RNA was processed using Illumina TotalPrep RNA Amplification Kit. Hybridization of human samples was performed on Illumina Human-HT12 Version 4.
Project description:Analysis of expression profile of peripheral blood from pancreatic ductal adenocarcinoma patients RNA expression profile of peripheral blood from pancreatic ductal adenocarcinoma patients Total RNA was isolated from peripheral blood. 36 patients with unresectable PDAC were recruited. The diagnosis of PDAC was based on clinical evaluation and imaging studies, which were histologically confirmed by surgery or imaging-guided biopsy. 14 gender, age, and habits matched healthy controls were also included. A total of 1000 ng of total RNA was processed using Illumina TotalPrep RNA Amplification Kit. Hybridization of human samples was performed on Illumina Human-HT12 Version 4.
Project description:This experiment describes the differential gene expression between parental gemcitabine-sensitive BxPC-3 cells and their resistant subclones, Bx-GEM. To select for the resistant subclones, parental BxPC-3 cells were treated with increasing concentrations of gemcitabine (10, 25, 50, 100 and 200 nM) for more than one year. Cells resistant at each stage of drug dosing were re-cultured in the subsequent dose, and their resistance confirmed via cell viability assays. Subclones resistant to 200 nM gemcitabine, in additon to the parental cells were used for gene profiling by array. Total RNA was extracted from the cells using the miRNeasy Mini Kit (Qiagen). The Illumina TotalPrep RNA Amplification Kit (life technologie) was used to generate single-stranded cRNA from input amounts of 500ng total RNA. 1,5 ug of cRNA were hybridized for 17 hr at 58°C on Illumina human HT12-Microarray using the Standard Illumina Hybridisation Protocol Part # 11322355 (Whole-Genome Gene Expression Direct Hybridization Assay Guide). Gene Expression Microarrays were scanned using the Illumina iScan-Scanner according to the Standard Illumina Scanning Protocol Part # 11322355 (Whole-Genome Gene Expression Direct Hybridization Assay Guide).
Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in iBAT obtained from Mettl3flox/flox and BKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from iBAT of Mettl3 flox/flox and BKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 5 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Chemically fragmented poly(A)+ RNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation following the standard protocol of Magna MeRIPTM m6A Kit (MERK, 17-10499). Enrichment of m6A mRNA was then analyzed by high-throughput sequencing using Illumina Hiseq X platform. The m6A peaks were detected by MACS2. The motif search was detected by HOMER. Conclusion: The iBAT mRNA m6A profiles in Mettl3flox/flox and BKO mice were characterized.
Project description:Ventricular myocytes were isolated from adult male Sprague-Dawley rats (250-350g) by collagenase digestion. Cells were cultured with recombinant human interleukin 6 for six hours prior to isolation total RNA by RNeasy mini kit (Qiagen). RNA was amplified and labelled using the Illumina TotalPrep RNA Amplification kit (Applied Biosystems) and hybridised to RatRef-12 Expression BeadChip (Illumina) according to manufacturer's instructions.
Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in the liver obtained from Mettl3flox/flox and HKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from liver of Mettl3 flox/flox and HKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 3 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Chemically fragmented poly(A)+ RNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation following the standard protocol of Magna MeRIPTM m6A Kit (MERCK, 17-10499). Enrichment of m6A mRNA was then analyzed by high-throughput sequencing using Illumina Hiseq X platform. The m6A peaks were detected by MACS2. The motif search was detected by MEME and DREME. Conclusion: The mRNA m6A profiles in the liver of Mettl3flox/flox and HKO mice were characterized.
Project description:Total RNA from 128 primary breast tumors was labelled using the Illumina TotalPrep RNA Amplification kit (Ambion) following manufacturers instructions. 1.5 ug of biotin-labelled cRNA were used for each hybridisation on Sentrix Human-6 BeadChips v1.0 (Illumina, San Diego, CA) following manufacturers protocol.
Project description:Total RNA from 46 breast cell lines was labelled using the Illumina TotalPrep RNA Amplification kit (Ambion) following manufacturer�s instructions. 1.5 �g of biotin-labelled cRNA were used for each hybridisation on Sentrix Human-6 v1 BeadChips (Illumina, San Diego, CA) following manufacturer�s protocol.
Project description:Total RNA from seven breast cell lines and one normal RNA samples was labelled using the Illumina TotalPrep RNA Amplification kit (Ambion) following manufacturer's instructions. 1.5 ug of biotin-labelled cRNA were used for each hybridisation on Sentrix Human-6 BeadChips (Illumina, San Diego, CA) following manufacturer's protocol.
Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in iBAT obtained from Wtapflox/flox and Wtap-BKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from iBAT of Wtap flox/flox and Wtap-BKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 8 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Fragmented mRNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs).The library preparations were sequenced on an Illumina Novaseq 6000 platform with a paired-end read length of 150 bp according to the standard protocols. The m6A peaks were detected by exomePeak R package (version 2.16.0). The motif search was detected by HOMER(version 4.9.1). Conclusion: The iBAT mRNA m6A profiles in Wtapflox/flox and BKO mice were characterized.