Dataset Information


The Involvement of mir-146 in Microglial Cells (EOC 13.31) During Prion Disease

ABSTRACT: MicroRNAs (miRNAs) are evolutionary conserved, non-coding, gene regulatory RNA molecules found in both plants and animals and amongst almost every cell and tissue type. They are about 22 nucleotides long and are involved in silencing of mRNA through sequence specific binding to the 3’ untranslated region (UTR) of the mRNA subsequently causing translational repression and/or will promote the degradation of protein-coding mRNA. Specifically, the miRNA family, mir-146 a/b, has been previously found to be involved in the regulation of the innate immune response by functioning as a negative regulator to help fine-tune the immune response. Microglial cells are the macrophage of the brain participating as major players of the innate immune response. During prion disease, no immune response is mounted against PrPSc possibly due to its similarity to host PrPc and thus, the host immune response would be suppressed and tightly regulated. Therefore, an increased expression of mir-146 by microglial cells during prion disease may function as one of these negative regulators. Our objective of this experiment is to use DNA microarrays to investigate the gene regulation of mir-146 previously found upregulated in our studies of mouse brain tissue specifically in microglial cells during prion disease with the aim of having a better understanding of prion pathobiology and a potential target for therapeutic intervention. Overall design: Mir-146 expression was confirmed via in situ analysis of brain tissue and was further investigated in a microglial murine cell line (EOC 13.31). To mimic mir-146 upregulation during prion disease, mir-146 a & b were overexpressed in EOCs using a lipid based reverse transfection system. Furthermore, endogeneous mir-146 was knocked down as another method to help confirm the mRNA targets affected by mir-146. Simultaneously, another experiment was performed for the investigation of its involvement in the innate immune response by stimulating the EOCs with differing concentrations of LPS from E.coli (055:B5). Total RNA was collected and prepared at several timepoints and the levels of expression of both mir-146 a & b was tested via qRT-PCR. The RNA collected from the EOCs from each condition were used as target material for dual-color, competitive hybridization to Agilent whole mouse genome 4x44K oligo arrays. All the significant targets found on the mircorarrays were compared against the various conditions to find a consensus of affected mir-146 mRNA targets.

INSTRUMENT(S): Agilent-014868 Whole Mouse Genome Microarray 4x44K G4122F (Feature Number version)

ORGANISM(S): Mus musculus  

SUBMITTER: Rhiannon LCH Huzarewich  




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