Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer, bladder cancer and renal cell carcinoma
Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer, bladder cancer and renal cell carcinoma
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DOI：
10.1111/j.1442-2042.2012.03082.x
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发表时间：
2012-11-01
影响因子：
2.6
通讯作者：
Ellinger, Joerg
中科院分区：
文献类型：
--
作者：
Sanders, Imke;Holdenrieder, Stefan;Ellinger, Joerg
目的：方法：收集前列腺癌（n = 24）、膀胱癌（n = 24）、肾细胞癌（n = 24）和正常对照组（n = 48）的血清，用cel-miR-39标记后，分离血清中的核糖核酸。使用定量实时聚合酶链反应来确定候选参考基因（RNU 1 -4、RNU 6 -2、SNORD 43、SNORD 44、SNORD 48、SNORA 74 A、miR-let-7a-1、miR-106 a）的水平。参考基因稳定性使用Normalization、geNorm和比较delta-Ct算法确定。用miR-21作为靶基因测试了正常化的效果，因为先前表明这在癌症患者的血清中上调。SNORD 44和SNORD 74 A水平在测定的检测限附近，因此被省略。所有剩余的候选基因显示出令人满意的稳定性;使用所有三种算法，SNORD 43是最稳定的参考基因。两个基因（SNORD 43、RNU 1 -4）的组合在一定程度上增加了稳定性。miR-21的水平是相似的癌症患者和健康对照组，无论正常化strategy.Conclusions：SNORD 43是一个合适的参考基因，用于分析循环微核糖核酸在泌尿系恶性肿瘤患者。我们的研究质疑miR-21作为泌尿肿瘤患者生物标志物的适用性。
Objectives: To identify an appropriate reference gene for the analysis of circulating micro-ribonucleic acid in patients with urological malignancies.Methods: Serum from patients with prostate cancer (n = 24), bladder cancer (n = 24), renal cell carcinoma (n = 24) and control subjects (n = 48) was spiked with cel-miR-39, and then ribonucleic acid was isolated. Quantitative real-time polymerase chain reaction was used to determine the levels of candidate reference genes (RNU1-4, RNU6-2, SNORD43, SNORD44, SNORD48, SNORA74A, miR-let-7a-1, miR-106a). Reference gene stability was determined using the NormFinder, geNorm and comparative delta-Ct algorithm. The effect of normalization was tested with miR-21 as the target gene, as this was previously suggested to be upregulated in cancer patients' serum.Results: Recovery of cel-miR-39 (mean 11.6%, range 156%) was similar in control subjects and cancer patients. SNORD44 and SNORD74A levels were around the detection limit of the assay and were thus omitted. All remaining candidates showed satisfying stability; SNORD43 was the most stable reference gene using all three algorithms. A combination of two genes (SNORD43, RNU1-4) increases the stability somewhat. The level of miR-21 was similar in cancer patients and healthy controls, irrespective of the normalization strategy.Conclusions: SNORD43 is a suitable reference gene for the analysis of circulating micro-ribonucleic acid in patients with urological malignancies. Our study questions the suitability of miR-21 as a biomarker for uro-oncological patients.