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Microarray Analysis of Injured Sciatic Nerves of WT and V30M Mice (Agilent Mouse Whole Genome 4x44k v2)


ABSTRACT: Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary disorder characterized by the extracellular deposition of mutant transthyretin (TTR). The most common TTR mutation in FAP results from an exchange of a methionine for a valine at position 30 – TTR V30M. In an attempt to establish an animal model of TTR Met-30-associated homozygous familial amyloidotic polyneuropathy and to study the structural and functional properties of human TTR Met 30, it was generated a mouse line carrying a null mutation at the endogenous ttr locus (ttr-/-) and the human mutant ttr gene (6.0-hMet 30) as a transgene. For microarray analysis, messenger RNA (mRNA) from WT and V30M injured sciatic nerves was extracted 7 days post-lesion (n=2 per strain), using the Lipid Tissue Mini Kit (Qiagen). mRNA integrity was assessed with Experion RNA StdSens Analysis Kit (Bio-Rad); samples used had a similar RNA quality indicator (RQI). Equal amounts of mRNA extract (200 ng) from each replicate were amplified and Cy-3-labeled using the Low Input Quick Amp Labeling kit (Agilent Technologies). In vitro synthesized transcripts (Spike-in kit), in pre-determinated ratios, were used to monitor microarray workflow for linearity, sensitivity and accuracy. Hybridizations were carried out at 65°C for 17 h, following Agilent manufacture’s instructions for One-Color Microarray-Based Gene Expression Analysis, using Mouse whole-genome 4 × 44K v2 Microarray slides. After washing, microarray slides were immediately scanned in the Agilent G2565AB microarray scanner and fluorescence quantification performed with Agilent Feature Extraction 10.5.1.1 software, following the GE1_105_Dec08 protocol. The signal intensity was aligned by centering the median of the signal distribution using the Biometric Research Branch (BRB)-ArrayTools v3.4.0, taking median array intensity as reference.

INSTRUMENT(S): Agilent G2565AB microarray scanner

ORGANISM(S): Mus musculus

SUBMITTER: Diana Raquel Fernandes Martins 

PROVIDER: E-MTAB-4525 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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