title: 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.
authors.affiliation: Department of Internal Medicine, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea.
authors.forename: Hye-Suk
authors.initials: HS
authors.lastname: Han
authors.affiliation: Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 363-883, Republic of Korea.
authors.forename: Yeong Nang
authors.initials: YN
authors.lastname: Jo
authors.affiliation: Public Health Medical Service, SMG-SNU Boramae Medical Center, Seoul 156-707, Republic of Korea.
authors.forename: Jin Yong
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Department of Pathology, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea.
authors.forename: Song-Yi
authors.initials: SY
authors.lastname: Choi
authors.affiliation: Department of Internal Medicine, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea.
authors.forename: Yusook
authors.initials: Y
authors.lastname: Jeong
authors.affiliation: Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 363-883, Republic of Korea.
authors.forename: Jieun
authors.initials: J
authors.lastname: Yun
authors.affiliation: Department of Pathology, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea.
authors.forename: Ok-Jun
authors.initials: OJ
authors.lastname: Lee
keywords: microRNAs; pleural effusion; reference