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

Stephen Chim,Ting-Fung Chan,Yvonne Cheng,Claire Chung,Stephanie Lam,Keun-Young Lee,Karen Wong,Oi-Ka Chan,Annie Hui,Chit-Ying Lai,Tak-Yeung Leung,Jamie Kwok,Stephen Tsui,Meng Meng

doi:10.3390/ijms18081709

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.

Highlights

Quantitative polymerase chain reaction is a standard method for the quantification of nucleic acid sequences [1,2]
To control for experimental error between samples that can be introduced at a number of stages throughout the procedure, the normalization of the reverse transcription (RT)-Quantitative polymerase chain reaction (qPCR) data is essential before analysis
To better understand spontaneous preterm birth (sPTB), using gene expression microarrays, panels of RNA transcripts were systematically identified to be aberrantly expressed in the placentas [7] and maternal blood cell samples [8] collected from pregnancies undergoing sPTB. Since these preterm birth-associated transcripts are detectable in maternal whole blood, in this study, we aimed to identify reference genes suitable for normalizing RNA transcripts in the whole blood of women undergoing preterm labor

Summary

Introduction

Quantitative polymerase chain reaction (qPCR) is a standard method for the quantification of nucleic acid sequences [1,2]. To control for experimental error between samples that can be introduced at a number of stages throughout the procedure, the normalization of the RT-qPCR data is essential before analysis. This is usually achieved by the use of a reference gene as an internal control that is presumed to remain relatively constant across different samples. It has been demonstrated that a proper choice of reference genes is highly dependent on the tissues or cells under investigation [4]. Reference genes are highly specific for a particular experimental model, and validation for each situation, on an individual basis, is a crucial requirement [5]

Objectives

Methods

Results