Identification of a suitable qPCR reference gene in metastatic clear cell renal cell carcinoma.

Piotr M Wierzbicki,Marcin Stanislawowski,Tomasz Slebioda,Marcin Matuszewski,Agata Wronska,Anna Kowalczyk,Zbigniew Kmiec,Agnieszka Rybarczyk,Jakub Klacz

doi:10.1007/s13277-014-2566-9

Piotr M Wierzbicki, Marcin Stanislawowski + Show 7 more

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https://doi.org/10.1007/s13277-014-2566-9

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Abstract

There is no data on reference gene (RG) selection in metastatic clear-cell renal cell carcinoma (mccRCC) for quantitative PCR (qPCR) data normalization. We aimed at selecting the most stable RG for further determination of new prognostic markers. Thirty-five nonmetastatic and 35 mccRCC patients undergoing radical nephrectomy were included. Paired primary tumor (T, n = 70) and normal (C, n = 70) kidney fragments were collected; from 12 out of 35 mccRCC cases, we also collected metastasized regional lymph nodes and adrenal gland tissues (M, n = 12). After RNA extraction, reverse transcription and qPCR were performed. Samples were divided into four analyzed groups. Fifteen candidate RGs were tested by RefFinder tool and manual statistics. To present the importance of RG selection, TP53 gene expression levels in samples were normalized with the use of RG data. RPL13 gene was the most stable RG in analysis of 35 primary tumor nonmetastatic versus 35 mccRCC samples and matched metastasized T/C/M samples (n = 12, each group). GUSB was the most suitable RG in total 152 samples and in paired T and C (n = 140) kidney samples. Expression of GUSB, RPL13, and the RPL13 + RPLP0 pair were independent of clinical/sample variables. Normalization of TP53 expression levels showed variability of GAPDH and ACTB assays. GUSB or RPL13 assays should be used in mccRCC for qPCR data normalization whereas GAPDH and ACTB assays should be avoided. Prior RG studies should precede each qPCR gene expression study since RG selection is associated with the origin and proportion of specimens.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-014-2566-9) contains supplementary material, which is available to authorized users.

Highlights

Renal cell carcinoma (RCC) is the third most common genitourinary malignancy, and its incidence has increased in the last 20 years [1]
Most clear-cell renal carcinoma (ccRCC) gene expression studies are normalized to GAPDH or ACTB genes [6], whose variable expression levels were noticed in other malignancies [8,9,10]
From 12 metastatic clear-cell renal cell carcinoma (mccRCC) patients, apart from tumor and kidney tissue, we collected metastasized tissue samples from regional lymph nodes and adrenal glands (n=10 and n=2, respectively) due to additional lymphadenectomy and/or adrenectomy performed during nephrectomy (Fig. 1); metastasized tissue specimens from remaining 23 mccRCC cases were not obtained for this study

Summary

Introduction

Renal cell carcinoma (RCC) is the third most common genitourinary malignancy, and its incidence has increased in the last 20 years [1]. The most frequent RCC subtype, clear-cell renal carcinoma (ccRCC) is characterized by a very high mortality rate of 40 %, due to distant metastases found in 30 % of RCC-diagnosed patients [2]. Expression level of a properly selected RG cannot be influenced by any clinical variable of the analyzed specimens/patients, i.e., sample origin, disease stage, or a pharmacological treatment [6, 7]. Most ccRCC gene expression studies are normalized to GAPDH or ACTB genes [6], whose variable expression levels were noticed in other malignancies [8,9,10]. The first aim of our study was to select the most stable RG among 15

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