Ohl Falk, Jung Monika, Radonić Aleksandar, Sachs Markus, Loening Stefan A, Jung Klaus
Department of Urology and Clinic of Internal Medicine II (Oncology and Hematology), Charité-University Medicine Berlin, Berlin, Germany.
J Urol. 2006 May;175(5):1915-20. doi: 10.1016/S0022-5347(05)00919-5.
Housekeeping genes as endogenous references are generally used for the relative quantification of target genes in gene profiling studies. To date that issue has not been sufficiently investigated in bladder cancer. From a panel of 9 potential candidates we selected the most stable housekeeping genes for gene normalization in bladder cancer tissue.
Expression profiles of the 9 genes ACTB, ALAS1, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, SDHA and TBP were established in matched malignant and nonmalignant tissue specimens from 14 patients with bladder cancer. Quality assessment of isolated RNA was performed with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California) and real-time reverse transcriptase-polymerase chain reaction was performed with LightCycler. The software geNorm and NormFinder (Aarhus University, Aarhus, Denmark) were used to identify the most suitable reference genes.
RNA was isolated with high purity and integrity. Candidate reference genes showed a broad range of between 20 and 34 polymerase chain reaction cycles. Expression did not depend on patient sex or tumor stage. GAPD, G6PD and HMBS showed significant differences in expression between malignant and nonmalignant pairs (at least p <0.04). Expression of the remaining genes did not differ between the matched pairs. SDHA and TBP were the most stably expressed genes, covering higher and lower expression levels.
For normalization purposes in gene profiling studies of bladder cancer the genes SDH and TBP are recommended as single reference genes depending on the expression level of the target gene or more favorably in combination.
管家基因作为内源性参照，通常用于基因谱研究中靶基因的相对定量分析。迄今为止，该问题在膀胱癌中尚未得到充分研究。我们从9个潜在候选基因中筛选出用于膀胱癌组织基因标准化的最稳定管家基因。
在14例膀胱癌患者配对的恶性和非恶性组织标本中，检测了9个基因（ACTB、ALAS1、G6PD、GAPD、HMBS、HPRT1、K-ALPHA-1、SDHA和TBP）的表达谱。使用2100生物分析仪（安捷伦科技公司，加利福尼亚州帕洛阿尔托）对提取的RNA进行质量评估，并使用LightCycler进行实时逆转录聚合酶链反应。使用geNorm和NormFinder软件（丹麦奥胡斯大学）来确定最合适的参照基因。
RNA提取的纯度和完整性高。候选参照基因的聚合酶链反应循环数在20到34之间变化较大。基因表达不依赖于患者性别或肿瘤分期。GAPD、G6PD和HMBS在恶性和非恶性配对组织间的表达有显著差异（至少p<0.04）。其余基因在配对组织间的表达无差异。SDHA和TBP是表达最稳定的基因，涵盖了较高和较低的表达水平。
在膀胱癌基因谱研究中进行标准化时，建议根据靶基因的表达水平，将SDH和TBP基因作为单一参照基因，或更优地将它们联合使用。
