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

Tim Erkens,Luc J Peelman,Karen Goossens,Jo Vandesompele,Mario Van Poucke,Alex Van Zeveren

doi:10.1186/1472-6750-6-41

Tim Erkens, Luc J Peelman + Show 4 more

Open Access

https://doi.org/10.1186/1472-6750-6-41

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Journal: BMC biotechnology	Publication Date: Oct 9, 2006
Citations: 199	License type: cc-by

Affiliation: Ghent University, Ghent University Hospital

Abstract

BackgroundAn 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.ResultsThe 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).ConclusionThis 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.

Highlights

An essential part of using real-time reverse transcription (RT)-PCR is that expression results have to be normalized before any conclusions can be drawn
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 first aim of this study was to develop a set of reference genes that can be used for normalizing real-time RT-PCR mRNA expression data from backfat and longissimus dorsi muscle of the pig

Summary

Introduction

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. 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. Two strongly genetically determined factors influencing meat quality are the amount of intramuscular fat (IMF) and muscle fibre type composition [2]. Meat quality increases if more oxidative fibres and less glycolytic fibres are present, but the influence of fat distribution and fibre type composition on meat quality varies between different breeds [1,5,6]

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