In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

Monika Jung¹ , Azizbek Ramankulov¹^,3 , Jan Roigas¹ , Manfred Johannsen¹ , Martin Ringsdorf¹ , Glen Kristiansen² and Klaus Jung¹

¹Department of Urology, Charité – Universitätsmedizin Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany

²Institute of Pathology, Charité – Universitätsmedizin Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany

³Republic Center of Urology, Bishkek, Kyrgyz Republic

author email corresponding author email

BMC Molecular Biology 2007, 8:47doi:10.1186/1471-2199-8-47


Published:	8 June 2007

Abstract

Background

Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes.

Results

The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel.

Conclusion

Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.