Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues

Uddin et al. BMC Research Notes 2011, 4:441 http://www.biomedcentral.com/1756-0500/4/441 RESEARCH ARTICLE Open Access Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues Muhammad Jasim Uddin1,2, Mehmet Ulas Cinar1, Dawit Tesfaye1, Christian Looft1, Ernst Tholen1 and Karl Schellander1* Abstract Background: 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. 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. Results: The 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. Conclusions: Although, 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. Background The pig is one of the most studied organism in research community as a food as well as a model animal, and many projects in pigs require the quantification of genes for many purposes. Real-time quantitative PCR (qRT-PCR) is the most frequently used method for gene quantification nowadays. qRT-PCR is an efficient method for quantification of mRNA transcript levels due to its high sensitivity, reproducibility and large dynamic range. Furthermore, it is * Correspondence: 1 Animal Breeding and Husbandry/Genetics group, Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany Full list of author information is available at the end of the article fast, easy to use and provides simultaneous measurement of gene expression in many different samples for a limited number of genes [1-3]. In case of qRT-PCR, when analyzing data for relative quantification, results are normalized to a reference. The most accepted approach to mRNA quantification is normalization of the expression level of a gene of interest (target gene) to the expression level of an internal stably expressed gene (control gene) [4-6]. The control gene, often termed reference gene or housekeeping gene, is a stably expressed gene that is experimentally verified in given species and tissues under given experimental conditions [3,7-9]. Normalizing to a reference gene is a widely used method because it is simple in theory. The © 2011 Schellander et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Uddin et al. BMC Research Notes 2011, 4:441 http://www.biomedcentral.com/1756-0500/4/441 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. However, this approach requires the selection of at least one reference gene for validation of a corresponding qRTPCR method. Normalization is extremely important to allow accurate comparison of the results between different samples and conditions in gene expression studies [4]. For instance, the commonly used reference genes such as GAPDH and b-actin are unfortunately 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 [10-12]. It is therefore necessary to validate the expression stability of reference genes prior to their use in an experimental protocol. Recently it has been recommended that a combination of reference genes should be used to obtain a more stable reference [6] and the use of a single reference gene is nowadays discouraged by more and more authors [4,6,13]. Because, a variability or alteration in the chosen reference gene by the experiment, however, may change the obtained results entirely and could be incorrect. Therefore, the validation of potential reference genes is essential. An ideal reference gene should be stably expressed and unaffected by experimental protocol or status [14]. But, the recent studies showed that the housekeeping gene expressions could be changed according to the type of tissues [3,8,15] breeds [15], experimental condition (such as treatment or disease) [16-19] and age [15,20,21]. A set of reference genes are suggested on the basis of their stability over tissues in pigs [3,7,15,22,23] but studies for expression stability of housekeeping genes in varieties of porcine tissue collected from different age of pigs are rare. Therefore, 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. Methods Tissues collection A total of nine clinically healthy pigs of three age group were selected: neonatal (one day old), young (2 months old) and adult (5 months old) for this experiment. Each age group (...truncated)