Combined analysis of multiple mRNA markers by RT-PCR assay for prostate cancer diagnosis Adriana F. Neves a, ⁎ , Thaise G. Araújo a , Weruska K.F.S. Biase a , Juliana Meola a , Tânia M. Alcântara b , Danielo G. Freitas c , Luiz R. Goulart la a Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil b Department of Pathology, Federal University of Uberlandia, Hospital de Clínicas of Uberlandia, Minas Gerais, Brazil c Urology Division, Federal University of Uberlandia, Hospital de Clínicas of Uberlandia, Minas Gerais, Brazil Received 4 April 2008; received in revised form 23 June 2008; accepted 24 June 2008 Available online 2 July 2008 Abstract Objectives: To develop a semi-quantitative method for prostate cancer diagnosis and to validate this technique in clinical protocols with the use of multiplex RT-PCR assays for five different biomarkers associated with carcinogenesis, including the PCA3 gene. Design and methods: AR, SRD5A2, KLK2, PSMA, and PCA3 transcripts were analyzed by multiplex RT-PCR assay in 73 prostatic tissue samples from patients with prostate cancer (PCa) and benign hyperplasia (BPH). Results: Significant differences were observed between cancerous and hypertrophic tissues in the relative expression of these genes. AR, KLK2, PSMA, and PCA3 genes displayed increased transcriptional levels in the cancer specimens; on the other hand, SRD5A2 mRNA levels were higher in the BPH samples. Conclusions: Our results suggest that the most promising marker for PCa diagnosis was positive PCA3 detection associated with serum PSA levels, which showed 28-fold higher chances for cancer occurrence, with 92% specificity and 94% positive predictive value. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Androgen receptor; 5-alpha reductase type 2; Human kallikrein 2; Prostate cancer antigen 3; Prostate-specific antigen; Prostate-specific membrane antigen; Reverse transcriptase polymerase chain reaction; Prostate cancer; Benign prostatic hyperplasia Introduction Testosterone is converted into dihydrotestosterone (DHT) by the 5-alpha reductase enzyme (SRD5A2) in prostatic cells, with consequent activation of genomic and proteomic pathways [1]. To understand the complex mechanisms associated with pros- tate cancer development, it is important to analyze the 5-alpha reductase enzyme at both the transcriptional and proteic levels [2,3]. DHT modulates gene transcription by association with the androgen receptor (AR) in the cytoplasm, acting in the nucleus by binding to androgen response elements (AREs) [1]. The androgen receptor is a member of a nuclear receptor family that transactivates gene expression and mediates the recruitment of co-activators and co-repressors [4–8]. Thus, the AR protein is considered a key molecule in the prostate cancer outcome, implying that its regulation could be at the transcrip- tional level. KLK2 and KLK3, members of kallikrein family, represent genes activated by the androgen receptor [9]. Kallikreins are a subgroup of serine proteases that play important roles in diverse physiological processes. The human kallikrein protein 2 (hK2), in vitro, activates the zymogen or the single-chain form of the urokinase-type plasminogen activator (uPA), and since uPA has been implicated in the promotion of cancer metastases, hK2 may be part of this pathway in prostate cancer [10,11]. The Available online at www.sciencedirect.com Clinical Biochemistry 41 (2008) 1191 – 1198 Abbreviations: AR, androgen receptor; BPH, benign prostatic hyperplasia; B2M, beta 2-microglobulin; KLK2/ hK2, human kallikrein 2; KLK3/ PSA, prostate-specific antigen; PCa, prostate cancer; PCA3, prostate cancer antigen 3; PSMA, prostate-specific membrane antigen; RT, reverse transcription; SRD5A2, 5-alpha reductase enzyme; tPSA, serum total PSA; TNM, tumor-node- metastasis; uPA, urokinase-type plasminogen activator. ⁎ Corresponding author. Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlandia, Campus Umuarama, Bloco 2E, Sala 24, CEP: 38400902, Uberlandia, Minas Gerais, Brazil. Fax: +55 34 3218 2203. E-mail address: neves.af@gmail.com (A.F. Neves). 0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.06.013