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Identification of suitable reference genes for the relative quantification of microRNAs in pleural effusion.

ABSTRACT: Circulating cell-free microRNAs (miRNAs) are potential biomarkers of cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is widely used in miRNA expression studies. The aim of this study was to identify suitable reference genes for RT-qPCR analyses of miRNA expression levels in pleural effusion. The expression levels of candidate reference miRNAs were investigated in 10 benign pleural effusion (BPE) and 10 lung adenocarcinoma-associated malignant pleural effusion (LA-MPE) samples using miRNA microarrays. The expression levels of candidate reference miRNAs, together with those of U6 small nuclear RNA (snRNA), RNU6B, RNU44 and RNU48 small RNAs, in 46 BPE and 45 LA-MPE samples were validated by RT-qPCR, and were analyzed using the NormFinder and BestKeeper algorithms. The impact of different normalization approaches on the detection of differential expression levels of miR-198 in BPE and LA-MPE samples was also assessed. As determined by the miRNA microarray data, five candidate reference miRNAs were identified. Following RT-qPCR validation, U6 snRNA, miR-192, miR-20a, miR-221, miR-222 and miR-16 were evaluated using the NormFinder and BestKeeper software programs. U6 snRNA and miR-192 were identified as single reference genes and the combination of these genes was preferred for the relative quantification of miRNA expression levels in pleural effusion. Normalization of miR-98 expression levels to those of U6 snRNA, miR-192 or a combination of these genes enabled the detection of a significant difference between BPE and LA-MPE samples. Therefore, U6 snRNA and miR-192 are recommended as reference genes for the relative quantification of miRNA expression levels in pleural effusion.

SUBMITTER: Han HS

PROVIDER: S-EPMC4156210 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Identification of suitable reference genes for the relative quantification of microRNAs in pleural effusion.

Han Hye-Suk HS Jo Yeong Nang YN Lee Jin Yong JY Choi Song-Yi SY Jeong Yusook Y Yun Jieun J Lee Ok-Jun OJ

Oncology letters 20140801 4

Circulating cell-free microRNAs (miRNAs) are potential biomarkers of cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is widely used in miRNA expression studies. The aim of this study was to identify suitable reference genes for RT-qPCR analyses of miRNA expression levels in pleural effusion. The expression levels of candidate reference miRNAs were investigated in 10 benign pleural effusion (BPE) and 10 lung adenocarcinoma-associated malignant pleural effusion (LA-M ...[more]

PMID: 25202432

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Project description:MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples.We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus,Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels.Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

Dataset Information

Identification of suitable reference genes for the relative quantification of microRNAs in pleural effusion.

Publications

Identification of suitable reference genes for the relative quantification of microRNAs in pleural effusion.

Similar Datasets

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