Systematic Selection of Reference Genes for the Normalization of Circulating RNA Transcripts in Pregnant Women Based on RNA-Seq Data
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AbstractRNA 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.
Acceptance Date29/07/2017
All Author(s) ListChim Stephen S C , Wong Karen K W, Chung Claire Y L , Lam Stephanie K W, Kwok Jamie S L, Lai Chit-Ying, Cheng Yvonne K Y, Hui Annie S Y, Meng Meng, Chan Oi-Ka, Tsui Stephen K W, Lee Keun-Young, Chan Ting-Fung, Leung Tak-Yeung
Journal nameInternational journal of molecular sciences
Volume Number18
Issue Number8
PublisherMDPI AG
Place of PublicationSwitzerland
LanguagesEnglish-United States
KeywordsNormFinder, Removal of Unwanted Variation from RNA-seq data (RUVSeq), blood biomarkers, denoise, geNorm, normalization, reference target, reverse-transcriptase polymerase chain reaction (RT-PCR), technical variation, transcriptomics

Last updated on 2021-08-06 at 01:17