GSE106550
Next Generation Sequencing Facilitates Quantitative Analysis of pancreatic tissue transcriptomes from Wild Type and miR-21-/- mice treated with caerulein
Purpose: Purpose: we used next generation sequencing to analyze gene expression profiles of pancreatic tissues from wild-type (WT) and miR-21-/- (miR-21 KO) mice treated with saline(control) or caerulein. The goals of this study are to compare the different gene expression profiles of pancreatic tissue between WT and miR-21 KO mice treated with caerulein. Methods: Female WT and miR-21 KO mice were administered 8-hourly intraperitoneal injection of 50µg/kg caerulein. Mice were sacrificed at 24 hours after the first caerulein injection and total RNA was extracted. mRNArna profiles were generated by deep sequencing WT treated with saline in single, WT treated with caerulein in duplicate, miR-21KO treated with caerulein in duplicate, using High-seq 2000 Illumina sequencing platform. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: After quality filtering of raw sequencing data, we obtained 32,797,327 (69.9%) out of 46,941,672 tags from WT mice treated with saline, 42,587,312 (87.1%) out of 48,919,213 tags from WT mice treated with caerulein, and 57,139,408 (85.1%) out of 67,111,415 tags from miR-21 KO mice treated with caerulein. We then mapped these tags to the mouse genome. We identified 1457 differential expressed genes (DEGs) between WT mice treated with caerulein and with saline, and 152 genes between WT mice and miR-21 KO mice treated with caerulein with a fold change more than 2.0 (up-regulation) or less than 0.5 (down-regulation). Altered expression of 16 genes was confirmed with qRT-PCR. Conclusions: Our study represents the first detailed analysis of pancreatic transcriptomes generated by RNA-seq technology. By using RNA-seq based transcriptome analysis, we identified 6 miR-21 target genes and 10 downregulated genes involved in pancreatic injury. Specifically, up-regulation of Pias3 and down-regulation of Hmgb1 when miR-21 was ablated coincided with reduced pancreatitis severity. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex pancreatic functions. Overall design: Pancreatic mRNA profiles of WT and miR-21 KO mice treated with caerulein were generated by deep sequencing, WT treated with saline in single, WT treated with caerulein in duplicate, miR-21KO treated with caerulein in duplicate, using High-seq 2000 Illumina sequencing platform.