Application of a Modified Real-Time PCR Technique for Relative Gene Copy Number Quantification to the Determination of the Relationship between NKX3.1 Loss and MYC Gain in Prostate Cancer

Abstract

Gene amplifications and deletions play an important role in the pathogenesis of solid tumors, including prostate cancer. Real-time PCR is a powerful tool for quantitative DNA analysis, particularly when starting quantities of tumor tissue are minimal (1)(2)(3)(4)(5). In the present study, we describe a modification of the 2−ΔΔCT method, which recently was shown to be suitable for relative gene expression analyses (6). We used our technique to analyze prostate cancer cell lines and tissue samples to determine the relationship between homeodomain-containing transcription factor 3.1 (NKX3.1) and MYC gene copy number alterations in this tumor type. The specimens analyzed included (a) blood samples from healthy donors; (b) the prostate cancer cell lines DU145 (ATCC no. HTB-81) and PC3 (ATCC no. CRL-1435) and their derived sublines DU145MN1, PC3-N, and PC3-125-1L (7); (c) the colorectal cell line COLO320DM (ATCC no. CCL-220), which harbors a high-level MYC amplification; and (d) primary prostate adenocarcinoma samples obtained after radical prostatectomy from previously untreated patients. The specimens were histologically verified, and samples were taken as described previously (8). We extracted DNA from the snap-frozen prostate carcinoma samples, blood samples, and cell lines, using the Blood and Cell Culture DNA Midi Kit (Qiagen). We performed quantitative real-time PCR using the LightCycler™ system (Roche Diagnostics) with the FastStart DNA Master SYBR Green I LightCycler Kit (Roche Diagnostics). PCRs were run in duplicate with 20-μL reaction volumes containing 1× SYBR Green I PCR Buffer Mix, 5 mM MgCl2, 0.5 μM each primer, 1× FastStart Taq DNA Polymerase (Roche Diagnostics), and 50 ng of genomic DNA. The cycling conditions are given in Table 1⇓ . The hot start PCR method was applied to prevent incomplete DNA denaturation as discussed by Wilhelm et al. (9). Melting curve analysis was performed …