Next Generation Sequencing Analysis of gene expression profile in E9.5 Mesp1Cre/+/Dgcr8-/- embryonic heart cells transfected with NC miRNA and miR-541 mimics
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ABSTRACT: Purpose: To compare the E9.5 Dgcr8 conditional knockout embryonic heart cells transfected with NC miRNA and miR-541 mimics Methods: In vitro cultured E9.5 Dgcr8 conditional KO heart cells transfected with miR-541-5p and NC miRNA were extracted with TRIZOL 48hrs after transfection, and 10ng total RNA was reverse transcribed and amplified by Smart-seq2 protocol as described (Picelli et al., 2014). Duplicated biological samples were analyzed using Illumina HiSeqX10, Clean reads were mapped to mouse genome (mm9) using BWA software. Results: Genes differentially expressed in E9.5 Dgcr8 cKO embryonic heart cells transfected with NC miRNA and miR-541 were identified. Conclusions: miRNA-541 significantly changes the gene expression profiles of E9.5 Dgcr8 cKO embryonic heart cells and promote the cardiac function
Project description:Purpose: To compare the transcriptome of wild type and Mesp1Cre+/Dgcr8-/- embryonic heart at embryonic stage E8.5 and E9.5 Methods: For mRNA analysis of embryonic heart during development, total RNA of E8.5 and E9.5 heart were extracted with TRIZOL, and 100ng total RNA was reverse transcribed and amplified by Smart-seq 2 protocol as described (Picelli et al., 2014). Duplicated biological samples were analyzed using Illumina HiSeq2500, Clean reads were mapped to mouse genome (mm9) using BWA software. Results: Genes significantly differentially expressed in the CKO cardiac tube at E9.5 compared with wild type were identified. Conclusions: Dgcr8 CKO caused important gene expression changes in E9.5 embryonic heart tube
Project description:Purpose: To compare the single cell transcriptome of wild type and Mesp1Cre+/Dgcr8-/- embryonic heart at embryonic stage E9.5 Methods: For single cell sample preparation, Ventricular part of E9.5 heart tubes were dissected and digested into single cells by 0.04% Trypsin combined with 0.05% Collagenase IV, and then transferred into DMEM containing 10% fetal bovine serum for termination. After washed in PBS without Ca2+, the single cells were manually transferred into cell lysis buffer with a mouth pipette. Total RNA was reverse transcribed and amplified using Smart-seq2 protocol (Picelli et al., 2014), followed by Tn5 tagmentation and PCR enrichment to generate libraries using TruePrep DNA Library Prep Kit V2 for illumina (Vazyme, TD501-503) according to the manufacturer’s suggested protocol. Clean reads were mapped to mouse genome (mm10) using Salmon. Results: Genes significantly differentially expressed in the E9.5 Dgcr8 cKO single cardiomyocyte compared with wild type group were identified. Conclusions: Dgcr8 cKO caused important gene expression changes in E9.5 cardiomyocytes at single cell level
Project description:Using an in vitro model of mouse corticogenesis from mouse embryonic stem cells (ESCs), we investigated the gene expression changes exerted by the inactivation of the Eutherian-specific microRNA miR-541. The microRNA mmu-miR-541-5p was inhibited by the transfection of an antago-miR into ESC-progenitors at 12 days of in vitro differentiation (DIV12) and and the cell transcriptome of transfected cells was compared to the transcriptome of control-transfected cells 5 days after transfection (DIV17). The comparison between control and miR-541 kd cells highlighted classes of differentially expressed genes related to axon elongation and neural development GO terms.
Project description:The goal of this study was to identify genes regulated by miRNA-29 in helper T cells. We compared gene expression in miRNA-deficient helper T cells (DGCR8-deficient) transfected with miR-29b versus control miRNA. We also compared gene expression in wildtype helper T cells transfected with miR-29 inhibitor to cells transfected with control inhibitor 3 biological replicates of each genotype (DGCR8-deficient or Wildtype C57BL/6 mice), with 2 conditions per genotype for 12 samples total. Cells from each DGCR8-deficient mouse were transfected with either miR-29b or control miRNA and cells from wildtype mice were transfected with either miR-29 inhibitors or control inhibitors
Project description:Purpose: To identify miRNA expresssion profiles in E9.5 mouse embryonic heart Methods: Total RNA of E9.5 heart were extracted with TRIZOL, miRNA deep sequencing were performed in using Illumina Hiseq 2500, SE50 (RIBOBIO, http://www.ribobio.com/), producing over 10 million reads from each sample. Clean reads were mapped to mouse genome (mm9), using miRDeep2 Results: MiRNAs that were highly expressed in E9.5 embryonic heart were identified Conclusions: Results provide insight into the role of miRNAs function in E9.5 embryonic heart development
Project description:The goal of this study was to identify genes regulated by miRNA-29 in helper T cells. We compared gene expression in miRNA-deficient helper T cells (DGCR8-deficient) transfected with miR-29b versus control miRNA. We also compared gene expression in wildtype helper T cells transfected with miR-29 inhibitor to cells transfected with control inhibitor
Project description:We performed Fluidigm C1 single cell sequencing analysis of wild-type and microRNA deficient (Dgcr8 knockout) mouse embryonic stem cells mock treated or transfected with either miR-294 or let-7.
Project description:let-7c and miR-294 were transfected into Dgcr8 -/- miRNA deficient ES cells and RNA was harvested after 12 hours the goal of this study was to identify direct and indirect targets of the let-7c and miR-294 miRNAs, we chose to harvest RNA early at 12 hours to mimize secondary effects due to ES cell differentiation, prior to performing the array experiment we found that at this time point candidate miRNA targets were maximally downregulated by qPCR
Project description:Three metastatic melanoma cell lines (BD-0548-ME, DP-0574-ME and WP-0614-ME) were transfected with miR precursors: hsa-miR negative control (NC) or hsa-miR-200a-3p precursors (miR-200a). The objective of this experiment was to determine miR-200a targets in metastatic melanoma cell lines. We selected low -miR-200a expression cell lines and then we overexpressed miR-200a or NC. Finally, we compared the metastatic melanoma cell lines with miR-200a overexpression (miR-200a) to the same cell line transfected with the negative control miR (NC).