Computational protocol: Is hepatic lipid metabolism of beef cattle influenced by breed and dietary silage level?

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Protocol publication

[…] Forward and reverse primers were optimally designed to cover exon–exon junctions to account for alternative splicing when possible (Table ).Primer 3 (http://primer3.ut.ee/) and Primer Express software v3.1 (Applied Biosystems, Foster City, CA, USA) were used to design primers for candidate housekeeping (HKG) and target genes, amplicon length was fixed to 70–150 bp. When possible, primer sets were designed to fall across exon–exon junctions. Sequences, amplicon length and reference sequences are summarised in Table . Primers were aligned against publicly available databases using BLASTN suite at the National Center of Biotechnology Information.Prior to RT-qPCR, the various sets of gene-specific primers were tested using a conventional PCR and run in a 2.5% agarose gel stained with ethidium bromide. Only those primers that did not present primer-dimer and a single band at the expected size in the gel were used for RT-qPCR. The accuracy of a primer pairs was also evaluated by the presence of a unique peak during the dissociation step at the end of RT-qPCR. A set of six candidate housekeeping genes was evaluated using geNorm and NormFinder, as described by Vandesompele et al.[] and Andersen et al.[], respectively. The target gene expression levels were calculated using the geometric mean of ribosomal protein S9 (RSP9) and succinate dehydrogenase complex subunit A (SDHA) as a normaliser.The efficiency of RT-qPCR amplification for each gene was calculated using the standard curve method with five dilutions at each data point along the curve. Dissociation curves were generated at the end of amplification to verify the presence of a single product. […]

Pipeline specifications

Software tools Primer3, Primer Express, BLASTN, NormFinder
Application qPCR
Organisms Bos taurus
Diseases Fatty Liver
Chemicals Fatty Acids, Triglycerides