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Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?

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Abstract

Using quantitative reverse transcription–polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of _target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and ≥7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the _target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.

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Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Ju 365/6-1/6-2 to K.J.) and by the Sonnenfeld-Stiftung Berlin (to K.J. and M.J.). This report includes parts of the doctoral thesis of F.O. We thank Britta Beyer for excellent technical assistance.

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Authors and Affiliations

  1. Department of Urology, Charité-Universitätsmedizin Berlin, Campus Mitte, Schumannstrasse 20/21, 10098, Berlin, Germany

    Falk Ohl, Monika Jung, Chuanliang Xu, Carsten Stephan, Anja Rabien, Stefan A. Loening & Klaus Jung

  2. Department of Urology, Changhai Hospital, Second Military Medical University, Shanghai, China

    Chuanliang Xu

  3. Institute of Pathology, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany

    Mick Burkhardt & Glen Kristiansen

  4. Robert Koch Institute, Berlin, Germany

    Andreas Nitsche

  5. Clinic of Internal Medicine II (Oncology and Hematology), Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany

    Aleksandar Radonić

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  1. Falk Ohl
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Ohl, F., Jung, M., Xu, C. et al. Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?. J Mol Med 83, 1014–1024 (2005). https://doi.org/10.1007/s00109-005-0703-z

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