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Int. J. Mol. Sci. 2017, 18(8), 1709; doi:10.3390/ijms18081709

Systematic Selection of Reference Genes for the Normalization of Circulating RNA Transcripts in Pregnant Women Based on RNA-Seq Data

1
Department of Obstetrics & Gynaecology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
2
School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
3
Hong Kong Bioinformatics Center, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
4
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
5
Department of Obstetrics & Gynaecology, Division of Maternal and Fetal Medicine, Kangnam Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07441, Korea
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 21 June 2017 / Revised: 27 July 2017 / Accepted: 29 July 2017 / Published: 4 August 2017
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
View Full-Text   |   Download PDF [1819 KB, uploaded 4 August 2017]   |  

Abstract

RNA transcripts circulating in peripheral blood represent an important source of non-invasive biomarkers. To accurately quantify the levels of circulating transcripts, one needs to normalize the data with internal control reference genes, which are detected at relatively constant levels across blood samples. A few reference gene candidates have to be selected from transcriptome data before the validation of their stable expression by reverse-transcription quantitative polymerase chain reaction. However, there is a lack of transcriptome, let alone whole-transcriptome, data from maternal blood. To overcome this shortfall, we performed RNA-sequencing on blood samples from women presenting with preterm labor. The coefficient of variation (CV) of expression levels was calculated. Of 11,215 exons detected in the maternal blood whole-transcriptome, a panel of 395 genes, including PPP1R15B, EXOC8, ACTB, and TPT1, were identified to comprise exons with considerably less variable expression level (CV, 7.75–17.7%) than any GAPDH exon (minimum CV, 27.3%). Upon validation, the selected genes from this panel remained more stably expressed than GAPDH in maternal blood. This panel is over-represented with genes involved with the actin cytoskeleton, macromolecular complex, and integrin signaling. This groundwork provides a starting point for systematically selecting reference gene candidates for normalizing the levels of circulating RNA transcripts in maternal blood. View Full-Text
Keywords: reference target; reverse-transcriptase polymerase chain reaction (RT-PCR); normalization; transcriptomics; blood biomarkers; geNorm; NormFinder; Removal of Unwanted Variation from RNA-seq data (RUVSeq); technical variation; denoise reference target; reverse-transcriptase polymerase chain reaction (RT-PCR); normalization; transcriptomics; blood biomarkers; geNorm; NormFinder; Removal of Unwanted Variation from RNA-seq data (RUVSeq); technical variation; denoise
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Chim, S.S.C.; Wong, K.K.W.; Chung, C.Y.L.; Lam, S.K.W.; Kwok, J.S.L.; Lai, C.-Y.; Cheng, Y.K.Y.; Hui, A.S.Y.; Meng, M.; Chan, O.-K.; Tsui, S.K.W.; Lee, K.-Y.; Chan, T.-F.; Leung, T.-Y. Systematic Selection of Reference Genes for the Normalization of Circulating RNA Transcripts in Pregnant Women Based on RNA-Seq Data. Int. J. Mol. Sci. 2017, 18, 1709.

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