NormFinder also enables estimation of the variation between sample subgroups, like

tumour and normal tissue, thus this algorithm can account for heterogeneity in the tested samples, which may be important considering the heterogeneity of the samples studied. The optimal normalization will vary with study design. The most suitable reference gene in one medical condition may be regulated in Gemcitabine mouse other tissues or diseases. Blanquicett et al., 2002, found that 18S, S9 and GUS were the least regulated genes among 15 putative reference genes when examining tumour and normal colorectal and liver tissues [28]. Furthermore, Dydensborg et al., 2006, identified B2M as the most appropriate gene for normalizing

colon carcinomas comparing to normal mucosa when they investigated seven colon adenocarcinomas containing both epithelial and stromal cells [29]. B2M was in this study identified as the least stable gene using NormFinder, and the third most variable gene using geNorm. In the present study where the tumour tissue samples consisted of more than 70% tumour cells some of the stromal cells are excluded. This might explain the discrepancies in the ranking of B2M since tumour tissue is heterogeneous and the fraction of INCB28060 manufacturer different cells may influence the gene expression results. Moreover, different patient groups, including age and clinical background, may also give dissimilarities across studies. CDK inhibitor Experimental variations may also influence the gene expression results, though using triplicates in the qRT-PCR analysis as used in this study will diminish this variation. Single assays qRT-PCR are time- and labour-intensive, 4��8C and require relatively large amounts of cDNA and PCR reagents in multivariate gene expression studies. TLDA overcome these drawbacks since this technique allows for simultaneously detection of expression of up to 384 genes and requires less template cDNA and PCR reagents than routine qRT-PCR [1, 31, 38–40]. Conclusions In this study we applied TaqMan Low Density Array in order to identify reference genes in

metastatic and non-metastatic colon cancer. The genes often used for normalization of gene expression data may be unstable and thus not suited for use, and therefore identifying stable reference genes in the specific experiment is vital for the results. The approach described herein can serve as a template to identify valid reference genes in any disease state. However, the optimal statistical approach to identify the best reference gene(s) remains to be determined. In the present study NormFinder and geNorm identified two different pairs of the most stable genes. The use of CTCV% might be a good validation of the two results. Nevertheless, the expression of target genes should be evaluated and a comparison of the effect of each pair of reference genes should be determined.