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Background  An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and  longissimus dorsi  muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition,  peroxisome proliferative activated receptor γ coactivator 1α  ( PPARGC1A ) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types.    Results  The mRNA expression stability of 10 reference genes was determined. The expression of  RPL13A  and  SDHA  appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes ( ACTB ,  TBP  and  TOP2B ), the results not only showed that the mRNA expression of  PPARGC1A  was significantly higher in each of the  longissimus dorsi  muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05).    Conclusion  This study provides a new set of reference genes ( ACTB ,  TBP  and  TOP2B ) suitable for normalization of real-time RT-PCR data of backfat and  longissimus dorsi  muscle in the pig. The obtained  PPARGC1A  expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.

bmc biotechnology

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A