Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Microarray analysis of genes impacted by 5 day knockdown of SLC24A5 in normal human melanocytes.

ABSTRACT: A major development in pigmentation research was the discovery of the involvement of SLC24A5 in natural variation in skin colour. This study used Agilent whole-genome microarrays to examine the impact of SLC24A5 knockdown on the transcriptome of normal human melanocytes derived from lightly and darkly pigmented donors. 2 melanocyte cell lines were treated for 6h, with 2 separate siRNA duplexes to target SLC24A5. Treatment of the cells with a non-targeting siRNA control duplex, lipofectamine transfection reagent alone and unchallenged cells, cultured alongside the cellular treatments comprised the controls for the experiment. All treatments and controls were conducted in triplicate, in both cell lines.

ORGANISM(S): Homo sapiens

SUBMITTER: Fei Ling Lim

PROVIDER: E-GEOD-21565 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes.

Shi Jing J Liu Meiqin M Shi Junna J Zheng Guangshun G Wang Yanping Y Wang Jinyu J Chen Yuzhen Y Lu Cunfu C Yin Weilun W

Plant cell reports 20120327 7

Ammopiptanthus mongolicus, the only evergreen broadleaf shrub endemic to the northwest desert of China, is a valuable species for plant abiotic stress research. No report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus. This work identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abc1, Ubc1, Ubc2, Ubc4, Ubc5, eIF1, eIF2, eIF3, ...[more]

PMID: 22451089

Publication: 1/3

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Project description:To provide insights into the role of Ankrd2 in the pathways controlling myogenic differentiation, the gene networks that are disturbed in Ankrd2 knockout or overexpressing primary cells were explored during differentiation. Ankrd2 plays an important role in myogenic differentiation by modulating the activity of its interacting partners (i.e p53). Since Ankrd2 binds to numerous transcription factors many signaling pathways remained to be explored to determine the functional significance of crosstalk between Ankrd2 and the activation of transcriptional programmes that regulate muscle remodeling. In contrast to previous gene expression studies in which we and others have studied the effect of Ankrd2 silencing in mouse differentiating C2C12 cells (Series GSE7542) and human differentiated CHQ5B muscle cells (Belgrano et al., 2011), we here used an in vivo experimental model in which the expression of Ankrd2 was completely abrogated. Furthermore, since Ankrd2 is known to be strongly upregulated in response to various stress stimuli, we studied the effect of Ankrd2 rescue and acute overexpression in KO and WT cells by adenoviral infection 20 hours prior to collection of cells. The pathways that change significantly are involved in distinct inflammatory response phases having NF-kB as a central node. Based on these results Ankrd2 is emerging for the first time as a repressor of immune and inflammatory responses. Gene expression profiling experiments have been performed using the SurePrint G3 Mouse 8x60K array by Agilent with 8 subarrays, each containing 39,430 60mer oligonucleotide probes for Entrez Gene RNAs, 16,251 for lincRNAs, 96 x 10 control probes, and 32 x 10 spike-in control probes. The Agilent platforms with one-color approach were selected to make possible the all-to all experiments comparison. The animal model, created and characterized in the laboratory of Dr.ML Bang, directed at knocking out Ankrd2 function was used. The adenovirus expressing Ankrd2-HA was produced using the AdEasy strategy (Agilent Technologies). Primary muscle cultures derived from wild type (WT) and Ankrd2-knockout (KO) mice infected with adenovirus expressing Ankrd2 or control GFP were collected at three different stages: proliferating, fusing and differentiated myoblasts. Three biological replicates per condition were included and a total of 36 microarray experiments were thus performed. First, the over-expression of Ankrd2 was checked by Western blot analysis. Then RNA was extracted from each sample and the relative transcriptional profiles were carried out using updated microarrays from Agilent technologies. Information from probe features was extracted from microarray scan data with the Feature Extraction Software (Agilent). Only arrays with at least 8/9 quality control metrics in range were used for the following data analyses. Intra-array normalization was performed with the Feature Extraction Software. Quantile inter-arrays normalization was performed using the Expander software (Sharan et al., 2003).

Project description:Mycobacterium bovis (M. bovis) causes bovine tuberculosis (bTB). The challenges in controlling and eradicating this zoonotic disease are compounded by our incomplete understanding of the host immune response. In this study, we used high-throughput bulk RNA sequencing (RNA-seq) to characterise the response profiles of γδ T cells to antigenic stimulation using purified protein derivate from M. bovis (PPDb). γδ T cells are a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. These cells are usually classified based on the expression of a pathogen-recognition receptor, Workshop Cluster 1 (WC1), into two main subsets: WC1.1+ and WC1.2+. Previous studies have identified a preferential transcriptomic response in WC1.1+ cells during natural bTB infection, suggesting a subset-specific response to mycobacterial antigens. This follow on study tested the hypothesis that a subset specific response would also be apparent from γδ T cells from infected cattle after repeat stimulation. Peripheral blood was collected from Holstein-Friesian cattle naturally infected with M. bovis, confirmed by a single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA and stimulated with 10 μg/ml PPDb for 6 hours. After whole blood stimulation, WC1.1+ and WC1.2+ γδ T cell subsets were isolated using magnetic cell sorting (n = 5 per group). High-quality RNA was extracted from each purified lymphocyte subset (WC1.1+ and WC1.2+) to generate transcriptomes using bulk RNA sequencing, resulting in 20 RNA-seq libraries. Transcriptomic analysis revealed 111 differentially expressed genes (DEGs) common to both WC1.1+ and WC1.2+ γδ T cell compartments, including upregulation of IL1A, IL1B, IL6, IL17A, IL17F, and IFNG genes (FDR-Padj. < 0.1). Interestingly, the WC1.2+ cells showed upregulation of IL10, CCL22, and GZMA (log2FC ≥ 1.5, and FDR-Padj. < 0.1). In conclusion, while WC1.1+ and WC1.2+ γδ T cells exhibit a conserved inflammatory response to PPDb, differences in anti-inflammatory and antimicrobial gene expression between these cell subsets provide new insights into their effector functions in response to mycobacterial antigens.

Dataset Information

Microarray analysis of genes impacted by 5 day knockdown of SLC24A5 in normal human melanocytes.

Publications

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes.

Similar Datasets

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