Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium

Sarah Arrowsmith

doi:10.1007/s11033-020-06066-2

Abstract

Accurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

Highlights

Real-time quantitative PCR is a sensitive method which enables the detection of small dynamic changes in mRNA levels and gene expression between different samples, for example to examine the effects of an experimental treatment or compare expression in tissue samples from two patient groups [1]
A total of 12 candidate reference genes (RGs) based on those used in previous studies of human myometrium and emerging RGs in other tissues types were used to identify the most suitable RGs for gene expression analysis using quantitative PCR (qPCR)
Exclusion of studies of myometrial pathology and non-human studies, as inferred from the article title, and review articles returned 189 items. Manual inspection of these articles resulted in the exclusion of 49 articles due to studies being in rodents which was not inferred from the title (n = 3), studies using other expression techniques and not qPCR (n = 25 including; ELISA, western blot, ChIP), semi-quantitative RTPCR (n = 9); RNA Seq (n = 3); meta-analysis of microarray and/or RNA Seq data (n = 2) and no RGs stated and which could not be inferred from the figures or list of primers used (n = 5)

Summary

Introduction

Real-time quantitative PCR (qPCR) is a sensitive method which enables the detection of small dynamic changes in mRNA levels and gene expression between different samples, for example to examine the effects of an experimental treatment or compare expression in tissue samples from two patient groups [1]. Many advances in the technological platforms in this field have arisen over recent years allowing for

Methods

Results

Discussion

Compliance with ethical standards