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Bulk RNA-seq of multi-tissue transcriptomes from control and DHEA-treated mice (ovary, uterus, thymus, brain, heart, intestine, kidney, liver and spleen)

ABSTRACT: To characterize systemic transcriptional changes induced by DHEA treatment, we performed bulk RNA-seq across a panel of tissues collected from control and DHEA-treated mice. Total RNA was extracted from each tissue using a kit-based workflow following mechanical homogenization in lysis buffer. RNA concentration/quality were assessed prior to library construction and high-throughput sequencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE316220 | GEO | 2026/01/16

REPOSITORIES: GEO

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Project description:ABSTRACT; The effects of DHEA were compared with those of DHT using gene expression array profiles in human LNCaP prostate cancer cells. LNCaP cells were exposed to DHEA (300 nM), DHT (300 nM), or vehicle for 48 hrs, and mRNA was isolated. mRNA expression was measured using Affymetrix HU-95 gene chips in 3 experiments performed on different dates. Gene expression values for the two treatment groups and control were sorted in ascending order on the p-values corresponding to a variance stabilized Hotelling test, which measured the extent of differential RNA expression between control and either hormone treatment. The top four genes with significant differential expression were S100 calcium binding protein, neurotensin, 24-dehydrocholesterol reductase, and anterior-gradient 2 homologue. Corresponding per comparison p-values were less than 3 x 10 -5. Nested tests of differential expression between DHEA and DHT treatment revealed significant differences (p < 0.01) for two of the four genes: the S100 calcium binding protein and neurotensin. The microarray findings were confirmed by quantitative RT-PCR. The top 83 genes found to exhibit differential expression were used in a pathway analysis. In general, DHT decreased expression of more genes involved in intercellular communication, signal transduction, nucleic acid binding and transport, and in structural components, such as myosin and golgin, than did DHEA. These data reveal consistent, measurable differences in gene expression patterns following treatment of LNCaP prostate cancer cells with DHEA versus DHT. The mechanisms underlying these observations, and the possible pathophysiological significance of these differences, remain to be determined. Experiment Overall Design: Three separate experiments were performed and labeled: 111502, 011403, and 022803. Three groups were run per experiment: Control, DHT or DHEA. Cells were exposed for 48 hr to 300 nM of DHT or DHEA. One Affymetrix HG_U95Av2 microarray chip per group was analyzed. The data was normalized by the "dchip" process.

Project description:The identification of putative biomarkers for predicting abdominal aortic aneurysm (AAA) progression can be achieved through proteomic strategies, particularly with shotgun proteomics. However, the heterogeneity of AAA tissue poses challenges for tissue homogenization and protein extraction, compromising reproducibility, an essential factor for clinical translation. Thus, we aimed to optimize a protocol for AAA tissue homogenization and protein extraction and to develop a standard operating procedure (SOP) to maximize protein yield and foster reproducibility in the field. In this context, several experimental variables were systematically tested in a bead-beating method for tissue homogenisation, including bead size (1.4 mm versus 2.8 mm of diameter), the number of extraction cycles (one up to three), a bead-to-tissue mass ratio at 20, 30, or 40, the lysis buffer-to-tissue ratio (10, 15, or 20 μL/mg), and the lysis buffer composition (RIPA, Urea/thiourea, and HEPES). Optimal conditions for protein extraction were achieved using 1.4 mm zirconium dioxide beads in two homogenization cycles, with a bead-to-tissue mass ratio of 30:1 and a lysis buffer-to-tissue ratio of 20 μL lysis buffer per milligram of tissue, in 2 mL O-ring cryotubes. Depending on the purpose of downstream analysis, recommendations for the use of each lysis buffers were provided based on their specific performance across specific indexes. In this context, HEPES and Urea/Thiourea buffers are recommended for studies prioritizing quantification reproducibility, while RIPA buffer is advised for protein extraction reproducibility. As a proof of concept, the optimized SOP was applied to characterize the AAA tissue proteome and several processes and pathways relevant in AAA pathophysiology were highlighted, including blood coagulation, cellular responses to stress, neutrophil degranulation, wound healing, and pathways of the immune system. Additionally, the enrichment analysis identified ECM remodeling and platelet activation, and degranulation as strongly represented pathways across the functional analysis for each buffer, reinforcing their contribution to aneurysm pathophysiology. Hence, these findings demonstrated that the SOP is well-suited for identifying disease-relevant biomarkers and therapeutic targets.

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Bulk RNA-seq of multi-tissue transcriptomes from control and DHEA-treated mice (ovary, uterus, thymus, brain, heart, intestine, kidney, liver and spleen)

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