Computational protocol: PPARα is essential for retinal lipid metabolism and neuronal survival

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Protocol publication

[…] Lipid transporter and FAO enzyme RNA levels were measured in WT vs. Pparα -/- retinas using quantitative real-time PCR (qRT-PCR) as described previously [], with minor modifications. Briefly, mice were euthanized with carbon dioxide asphyxiation and retinal RNA was isolated using a Qiazol (Qiagen, Valencia, CA, USA) extraction, purified using a PureLink® RNA Mini Kit (Qiagen) and treated with DNase I (Qiagen) to remove contaminating genomic DNA. DNase-treated RNA was then converted into cDNA using iScript® reverse transcriptase (BioRad, Hercules, CA, USA). PCR primers for target genes and housekeeping gene β-actin were designed using NCBI Primer Blast and OligoCalc software. Primer sequences are shown in Table . Quantitative analysis of gene expression was conducted using a BioRad CFX96® system with a SYBR Green Master Mix kit (BioTools, Jupiter, FL, USA) and target gene expression was calculated relative to β-actin using the ∆cT method. For in vivo studies, N is expressed as the number of retinas per genotype. For in vitro studies, N is expressed as the number of wells per group. […]

Pipeline specifications

Software tools Primer-BLAST, OligoCalc, Biotools
Applications Population genetic analysis, qPCR
Organisms Mus musculus
Diseases Macular Degeneration, Retinal Degeneration, Retinitis, Neurodegenerative Diseases
Chemicals Adenosine Triphosphate, Fatty Acids