Abstract
BackgroundGene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes or internal control genes is required. The stability of reference genes has a tremendous effect on the results of relative quantification of gene expression by qRT-PCR. The expression stability of reference genes could vary according to tissues, age of individuals and experimental conditions. In the pig however, very little information is available on the expression stability of reference genes. The aim of this research was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in varieties of porcine tissues at different ages.ResultsThe mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined in varieties of tissues collected from newborn, young and adult pigs. geNorm, NormFinder and BestKeeper software were used to rank the genes according to their stability. geNorm software revealed that RPL4, PPIA and YWHAZ showed high stability in newborn and adult pigs, while B2M, YWHAZ and SDHA showed high stability in young pigs. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that TBP was the most stable gene in newborn and young pigs, while PPIA was most stable in adult pigs. Moreover, geNorm software suggested that the geometric mean of three most stable gene would be the suitable combination for accurate normalization of gene expression study.ConclusionsAlthough, there was discrepancy in the ranking order of reference genes obtained by different analysing software methods, the geometric mean of the RPL4, PPIA and YWHAZ seems to be the most appropriate combination of housekeeping genes for accurate normalization of gene expression data in different porcine tissues at different ages.
Highlights
Gene expression analysis using real-time RT-PCR is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR
The commonly used reference genes such as GAPDH and b-actin are often used without prior validation of their expression stability under the specific study conditions, but a number of studies have shown that the expression of those genes is significantly altered in some experimental conditions [10,11,12]
This study was aimed to explore the expressions of nine mostly used housekeeping genes in 14 different tissues collected from three different ages of pigs (1 day old piglet, 2 months old young and 5 months old adult pigs) in order to select the suitable set of housekeeping genes that could be used as an internal control to normalize gene expression in pigs
Summary
Gene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. QRT-PCR is an efficient method for quantification of mRNA transcript levels due to its high sensitivity, reproducibility and large dynamic range It is normalization adjusts for differences in the quality or quantity of template RNA or starting material and differences in RNA preparation and cDNA synthesis, since the reference gene is exposed to the same preparation steps as the gene of interest. This allows the direct comparison of normalized transcript expression levels between samples. This approach requires the selection of at least one reference gene for validation of a corresponding qRTPCR method.
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