Computational protocol: Effect of the feeding system on the fatty acid composition, expression of the Δ9-desaturase, Peroxisome Proliferator-Activated Receptor Alpha, Gamma, and Sterol Regulatory Element Binding Protein 1 genes in the semitendinous muscle of light lambs of the Rasa Aragonesa breed

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Protocol publication

[…] Gene expression was analyzed by real-time RT-PCR (ABI Prism 7500 sequence detection system, Applied Biosystems, Madrid, Spain). According to the ovine SCD, PPARγ and PPARα cDNA sequences (GenBank Acc. Number AJ001048, AY137204, FJ200440 for SCD, PPARγ and PPARα respectively), primers for real-time RT-PCR were designed using the program Primer3 http://frodo.wi.mit.edu/primer3/. The sequences of the primers are shown in Table . The primers for SCD and PPARγ were designed across exon 4- exon 5 junction, while the primers for PPARα are designed in exon 7.However, no ovine SREBP1 gene sequences have been deposited in GenBank. Primers designed from bovine genomic DNA (GenBank Acc. Number NC_007317.3) were used to obtain partial genomic DNA regions of ovine SREBP1. Genomic DNA was amplified in a final volume of 25 μl containing 5 pmol of each primer 5'-CACTTCATCAAGGCAGACTC-3' and 5'-GAGCTCAAGGAGACTGGTGGT-3', 200 nM dNTPs, 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100 and 0.5 U Taq polymerase (Taq, Biotools). PCR amplification conditions included an initial denaturation step of 94°C for 3 minutes, then 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and then 72°C for 30 seconds. PCR products were sequenced using an ABI Prism 3700 (Applied Biosystems) and standard protocols. Sheep-specific primers were designed for real-time RT-PCR (Table ). The primers for SREBP1 real time RT- PCR are designed across exon 6- exon 7 junction. Homology searches were performed with BLAST (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/BLAST/), confirming the identity of the amplified fragment. Sheep-specific primers and the conditions for real-time RT-PCR are shown in Table .Before performing the real-time RT-PCR reactions, a conventional PCR was carried out for SCD, SREBP1, PPARα and PPARγ genes with the aim to test the primers and verify the amplified products. PCR products were sequenced to confirm the gene identity using an ABI Prism 3700 (Applied Biosystems) and standard protocols.The PCR was carried out in a total of 10 μl PCR mixture, containing SYBR Green PCR Master Mix (Applied Biosystems). Each reaction was run in triplicate and the average was used to calculate the relative amount of the target gene. Four housekeeping genes ovine beta actin (ACTB), succinate dehydrogenase (SDHA), glucose-6-phosphate dehydrogenase (G6PDH) and ubiquitin (UBC) stability were tested using geNorm software version 3.4., which calculates the measure of gene expression stability (M) of a putative reference gene based on the average pairwise variation between all investigated reference genes. The most stable genes were ACTB (M = 0,82) and G6PDH (M = 0,82) and the less stable UBC (M = 1,4). The geometric mean of the 3 most stable housekeeping genes were used to normalize each set of results ACTB, G6PDH and SDHA (M= 1,18) (4).The relative gene expressions were normalized against a factor that was based on the geometric mean of the expression levels of the three housekeeping genes, according to the recommendation of Vandesompele et al. []. The amplification conditions were an initial step of 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 59°C for 30 s. The specificity of the amplification products was determined using a melting curve in all cases. The efficiency of the PCR amplification for each gene was calculated using the standard curve method (E= 10-1/slope -1). The standard curves for each gene were generated by a five-fold serial dilution of pooled cDNA. Standart curve method was used to quantify the relative gene expression []. Normalized qPCR data were transformed in fold- change relative to group ALF. Normalized qPCR data were transformed to obtain a perfect mean of 1 in ALF group. PCR-normalized data are presented as n-fold change relative. […]

Pipeline specifications

Software tools Primer3, Biotools
Applications Population genetic analysis, qPCR
Organisms Ovis aries, Homo sapiens
Chemicals Linoleic Acids, Oleic Acid