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Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients.
BMC Urology ( IF 1.7 ) Pub Date : 2020-02-04 , DOI: 10.1186/s12894-019-0542-9
Sinisa Simonovic 1, 2, 3 , Christian Hinze 3 , Kai M Schmidt-Ott 3, 4 , Jonas Busch 1 , Monika Jung 1 , Klaus Jung 1, 2 , Anja Rabien 1, 2
Affiliation  

BACKGROUND RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort.

中文翻译:

基于qPCR的检测透明细胞肾细胞癌患者全血中肿瘤特异性循环mRNA的用途有限。

背景技术RNA测序数据提供了关于各种肿瘤中基因失调水平的丰富信息。这些数据以及基于较早的微阵列技术的数据已使人们能够鉴定与匹配的正常组织相比在透明细胞肾细胞癌(ccRCC)中上调的许多基因。在这里,我们使用RNA测序数据来构建ccRCC中一组高度过表达的基因,以便评估它们在全血中的RNA水平,并确定这些水平对肾细胞癌患者的诊断潜力。方法使用TCGA,GEO和其他数据库通过Python进行生物信息学分析,以鉴定ccRCC中上调而健康个体血液中不存在的基因。随后使用实时定量PCR(RT-qPCR)来测量16例ccRCC患者和11例健康个体的全血中候选基因(PAX基因)的水平。PCR结果在qBase和GraphPadPrism中进行处理,并使用Mann-Whitney U检验进行统计。结果尽管大多数分析的基因要么无法检测到,要么未显示任何表达失调,但与健康对照组相比,ccRCC患者血液中的两个基因CDK18和CCND1异常地被下调。此外,与非转移性相比,LOX在转移性ccRCC样品中显示出上调的趋势。结论该分析说明了检测血液中肿瘤调节基因的困难,以及即使对于正常血液中有条件缺乏的基因,血细胞表达干扰的可能影响。血浆样品中的测试表明,无法检测到肿瘤特异性mRNA。尽管CDK18,CCND1和LOX mRNA可能具有生物标志物的潜力,但这将需要在独立的较大患者队列中进行验证。
更新日期:2020-04-22
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