Abstract
BackgroundReproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.ResultsThe gene expression stability of ten potential reference genes was studied by three different methods (geNorm, NormFinder and BestKeeper) in ovary and uterus collected at five different physiological time points (heat, and 15, 30, 45 and 60 days of pregnancy). Although final ranking differed, the three algorithms gave very similar results. Thus, the most stable genes across time were TBP and UBC in uterus and TBP and HPRT1 in ovary, while HMBS and ACTB showed the less stable expression in uterus and ovary, respectively. When studied as a systematic effect, the reproductive stage did not significantly affect the expression of the candidate reference genes except at 30d and 60d of pregnancy, when a general drop in expression was observed in ovary.ConclusionsBased in our results, we propose the use of TBP, UBC and SDHA in uterus and TBP, GNB2L1 and HPRT1 in ovary for normalization of longitudinal expression studies using quantitative PCR in sows.
Highlights
Reverse-transcription quantitative PCR is a wellestablished method for estimating quantities of mRNA sequences
The main drawback of relative quantitation remains in the selection of an appropriate reference gene, whose expression levels would not be affected by the experimental conditions
BestKeeper 0.955 0.878 0.914 0.874 0.957 0.913 0.889 0.979 0.881 0.990 range of 18–24 except for HPRT1 and hydroxymethylbilane synthase (HMBS), which were less abundant than the rest (Table 1)
Summary
Reverse-transcription quantitative PCR (qPCR) is a wellestablished method for estimating quantities of mRNA sequences. It has greater sensitivity than other quantitation methods [1] and application of relative quantitation protocols eliminate the need of standards with known target concentrations In this type of study it is critical to normalize the amount of starting material with the use of one or more internal reference genes, which, ideally, should be present at constant levels across all samples in the experiment, at approximately equal concentration and amplified with equal efficiency as the target sequence [2]. The main drawback of relative quantitation remains in the selection of an appropriate reference gene, whose expression levels would not be affected by the experimental conditions This problem is especially relevant when comparing expression across a wide spectrum of tissues or when studying one tissue at different physiological or developmental stages. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages
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