Ohl Falk, Jung Monika, Xu Chuanliang, Stephan Carsten, Rabien Anja, Burkhardt Mick, Nitsche Andreas, Kristiansen Glen, Loening Stefan A, Radonić Aleksandar, Jung Klaus
Department of Urology, Charité-Universitätsmedizin Berlin, Campus Mitte, Schumannstrasse 20/21, 10098 Berlin, Germany.
J Mol Med (Berl). 2005 Dec;83(12):1014-24. doi: 10.1007/s00109-005-0703-z. Epub 2005 Oct 7.
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 >or=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.
在基因表达谱研究中，使用定量逆转录-聚合酶链反应（RT-PCR）时，参考基因被用作内源性对照，用于对靶基因进行相对定量。迄今为止，管家基因在前列腺癌组织中用于该目的的适用性尚未得到充分研究。本研究的目的是从16个潜在的候选参考基因中筛选出最稳定的基因用于基因标准化。通过实时RT-PCR检测了从17例未经治疗的前列腺癌根治术后获得的配对的、显微切割的恶性和非恶性组织标本中，编码ACTB、ALAS1、ALB、B2M、G6PD、GAPD、HMBS、HPRT1、K-ALPHA-1、POLR2A、PPIA、RPL13A、SDHA、TBP、UBC和YWHAZ的mRNA的表达。所研究的基因显示出广泛的表达范围，循环阈值在16至37之间。pT2和pT3肿瘤样本之间或Gleason评分<7和≥7的样本之间的表达无差异（P>0.05）。ACTB、RPL13A和HMBS在恶性和非恶性配对之间的表达存在显著差异（至少P<0.02）。所有其他基因在配对样本之间无差异，使用geNorm和NormFinder软件程序从这些候选基因中确定最合适的参考基因。这两个程序均计算得出HPRT1、ALAS1和K-ALPHA-1是表达范围广泛的最稳定基因。给出了单独用HPRT1以及用这三个参考基因组合产生的标准化因子以及不稳定基因ACTB或RPL13A对靶基因RECK进行标准化后的表达情况。该示例显示了在前列腺癌基因表达谱研究中使用合适的参考基因以避免错误标准化的重要性。在我们的研究中，单独使用HPRT1作为参考基因就足够了，但为提高前列腺癌基因表达谱研究中标准化的可靠性，应考虑由两个基因（HPRT1、ALAS1）或所有三个基因（HPRT1、ALAS1、K-ALPHA-1）产生的标准化因子。
