In the present study the expression stability and expression level of nine potential reference genes have been compared in 17 different pig tissues. Recently a set of reference genes ( ACTB, TBP and topoisomerase (DNA) II beta ( TOP2B)) have proven suitable for normalisation of qPCR data of backfat and longissimus dorsi muscle in the pig. The normalisation adjusts for differences in amount and quality of 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. The accurate quantification of reference genes allows normalisation of differences in amount of the amplified cDNA in individual samples. To control these variables, which are not a result of the experimental design, normalisation using reference genes are routinely used e.g. However, this does not take the RNA quality and enzymatic efficiencies into account. One way to standardize samples is to quantify the starting material i.e. One of the critical steps in comparing transcription profiles is accurate normalisation, therefore, a number of variables should be controlled such as amount of starting material, enzymatic efficiencies, differences in transcriptional activity and presence of inhibitors in different sample materials. Real-time quantitative PCR (qPCR) is an efficient method for quantification of mRNA transcription levels due to its high sensitivity, reproducibility and large dynamic range in addition, real-time qPCR is fast, easy to use and provides simultaneous measurement of gene expression in many different samples for a limited number of genes. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. ConclusionД®xpression stability varies greatly between genes. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/ RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH. The stability of these reference genes in different pig tissues was investigated using the geNorm application. The genes included beta-actin ( ACTB), beta-2-microglobulin ( B2M), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), hydroxymethylbilane synthase ( HMBS), hypoxanthine phosphoribosyltransferase 1 ( HPRT1), ribosomal protein L4 ( RPL4), succinate dehydrogenase complex subunit A ( SDHA), TATA box binding protein ( TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide ( YWHAZ). In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription.

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