Computational protocol: The influence of feeding linoleic, gamma linolenic and docosahexaenoic acid rich oils on rat brain tumor fatty acids composition and fatty acid binding protein 7 mRNA expression

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Protocol publication

[…] Total RNA was extracted from tumor samples using RNeasy mini kit as per manufacturer's instructions. RNA extract was analyzed spectrophometrically to determine purity and concentration and electrophoresd to check integrity. cDNA was synthesized from extracted RNA using QuantiTect Reverse Transcription Kit in which genomic DNA is removed by genomic DNA wipeout buffer before RT reaction. Synthesized cDNA was analyzed spectrophotometrically to determine concentration and stored in aliquots at -20°C until use.Table summarized primers used in this study. Primer sequence for PPARγ [], EGFR [] and RXRα [] and FABP7 [] were obtained from previous studies. Primer sequence for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Beta-Actin were designed by PerlPrimer [] and OligoAnalyzer (Integrated DNA Technologies), and the specificity of the sequences was analyzed by the BLAST database. Except for GAPDH, the primers were either located in different exons or across exon-exon boundaries.Real-time PCR was performed on a Corbett RotorGene 6000 (Corbett Research, Australia) using Quantifast SYBR-green PCR Master Mix. The PCR reactions contained 5 μL of 2X SYBR master mix, 250 ng of cDNA, and 300 nM of GAPDH, Beta-Actin, FABP7, RXR-α primers or 1 μM of EGFR or PPARγ primers in total volume of 10 μL and samples were run in duplicate. Thermal cycler conditions included holds for 5 minutes at 95°C, followed by 43 cycles of 15 seconds at 95°C and 40 seconds at 60°C.A melting curve analysis was performed for each reaction with 62–95°C ramp. No template control (NTC) consisting of H2O for target and reference genes were included in each run. Initially, electrophoresis on agarose gel were also performed to demonstrate that qRT-PCR yielded a unique band. The amplification efficiencies of target and reference genes were determined by dilution method, using 3 concentrations of cDNAs of 100, 150 and 250 ng for each gene. Three standard curve of three combined cDNAs of samples were drawn separately and mean efficiency for each gene determined and used for data analysis by Relative expression software tool (REST) []. Beta-actin and GAPDH served as reference genes. […]

Pipeline specifications

Software tools PerlPrimer, OligoAnalyzer, REST
Application qPCR
Organisms Rattus norvegicus, Carthamus tinctorius
Diseases Amino Acid Metabolism, Inborn Errors, Brain Neoplasms, Glioma, Neoplasms
Chemicals Fatty Acids, 8,11,14-Eicosatrienoic Acid, gamma-Linolenic Acid, Linoleic Acid