Computational protocol: Differential effects of short-term β agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle

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Protocol publication

[…] The relative expression of the adult MyHC isoforms (MyHCI, IIA, IIX and IIB) were determined at both the mRNA and protein level, as described previously (Hemmings et al., ). Briefly, SDS–PAGE separation of MyHC isoforms was carried out to determine percentage MyHC protein expression, comparing samples to a bovine positive control (Longissimus thoracis) sample (kindly provided by Dr Brigitte Picard, INRA, France) previously shown to express all four adult MyHC isoforms (Picard and Cassar-Malek, ). Quantitative real-time PCR was carried out on muscle cDNA to determine percentage MyHC mRNA expression, using ovine specific primers and probes for MyHCI, IIA and IIX (Supplementary Table S1). The relative percentage of each isoform was calculated as described previously (Hemmings et al., ). Primers and probes designed to the available sequence for the ovine MyHCIIX isoform would be expected to generate amplicons from any MyHCIIB transcripts present as well, as this region is identical in the porcine sequences for MyHCIIX and IIB, therefore expression is stated as MyHCIIX/IIB.The mRNA expression of the MyHCIIB isoform was analysed by semi-quantitative RT-PCR using published primers previously described as detecting only ovine MyHCIIB (Vuocolo et al., ). MyHCIIB expression was compared with the semi-quantitative RT-PCR analysis of MyHCIIX+IIB combined (using the primers in Supplementary Table S1). […]

Pipeline specifications

Software tools Picard, MUSCLE
Application Nucleotide sequence alignment
Organisms Ovis aries
Chemicals Lactic Acid