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

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.

SUBMITTER: Wierzbicki PM

PROVIDER: S-EPMC4275580 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Kidney renal clear cell carcinoma (KIRC) is the malignancy originated from the renal epithelium, with a high rate of distant metastasis. Aberrant alternative splicing (AS) of pre-mRNA are widely reported to be involved in the tumorigenesis and metastasis of multiple cancers. The aim of this study is to explore the mechanism of alternative splicing events (ASEs) underlying tumorigenesis and metastasis of KIRC. Methods: RNA-seq of 537 KIRC samples downloaded from the TCGA database and ASEs data from the TCGASpliceSeq database were used to identify ASEs in patients with KIRC. The univariate and Lasso regression analysis were used to screen the most significant overall survival-related ASEs (OS-SEs). Based on those, the OS-SEs model was proposed. The interaction network of OS-SEs and splicing factors (SFs) with absolute value of correlation coefficient value >0.750 was constructed by Pearson correlation analysis. The OS-SEs significantly related to distant metastasis and clinical stage were identified by non-parametric test, and those were also integrated into co-expression analysis with prognosis-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified by Gene Set Variation Analysis (GSVA). ASEs with significance were selected for multiple online database validation. Results: A total of prognostic 6,081 overall survival-related ASEs (OS-SEs) were identified by univariate Cox regression analysis and a prediction model was constructed based on 5 OS-SEs screened by Lasso regression with the Area Under Curve of 0.788. Its risk score was also illustrated to be an independent predictor, which the good reliability of the model. Among 390 identified candidate SFs, DExD-Box Helicase 39B (DDX39B) was significantly correlated with OS and metastasis. After external database validation, Retained Intron of Ras Homolog Family Member T2 (RHOT2) and T-Cell Immune Regulator 1 (TCIRG1) were identified. In the co-expression analysis, overlapped co-expression signal pathways for RHOT2 and TCIRG1 were sphingolipid metabolism and N-glycan biosynthesis. Conclusions: Based on the results of comprehensive bioinformatic analysis, we proposed that aberrant DDX39B regulated RHOT2-32938-RI and TCIRG1-17288-RI might be associated with the tumorigenesis, metastasis, and poor prognosis of KIRC via sphingolipid metabolism or N-glycan biosynthesis pathway.

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

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