Selection of Endogenous Control Reference Genes for Studies on Type 1 or Type 2 Endometrial Cancer

Thangesweran Ayakannu,Justin C Konje,Anthony H Taylor

doi:10.1038/s41598-020-64663-4

Abstract

A panel of 32 candidate reference genes was used to identify the most stable genes for gene normalisation in quantitative RT-PCR studies using endometrial biopsies obtained from women with endometrial cancer (type 1 or type 2) and without cancer (controls). RNA from the biopsies was isolated, examined for purity and quality, and then reverse transcribed into cDNA before being subjected to real-time qRT-PCR analysis in triplicate within the TaqMan gene Expression Assay kit. The most ‘stable’ endogenous control genes were then identified using the geNorm qbase + 2 and NormFinder software packages. PSMC4, PUM1 and IPO8 were identified as the best reference genes combination for type 1 endometrial cancer (grades 1, 2 and 3), whereas for type 2 endometrial cancer (serous and carcinosarcoma), UBC, MRPL19, PGK1 and PPIA were the best reference genes combination. We conclude that the use of these normaliser combinations should provide accurate interpretation of gene expression at the transcript level in endometrial cancer studies especially for types 1 and 2 cancers.

Highlights

Elimination of sources of error in the quantitative real-time PCR (qRT-PCR) technique, such as differences in the quantity and quality of extracted mRNA, the presence of contaminating genomic or operator DNA, divergences in reverse transcription and PCR efficiencies must occur for the qRT-PCR to be valid[16,17,18]
We showed that a combination of 3 genes (IPO8, MRPL19 and PPIA) provided the best combination of normalisation factors in qRT-PCR studies of endometrial cancer and that geNorm qbase+226 is more robust than the other software packages in its statistical corrections for all the possible sources of experimental error listed above
Using geNorm, the least stable to most stable reference genes evaluated for type 1 EC was found to be: RPL30 > MT-ATP6 > 18S > ACTB > TBP > RPLP0 > PES1 > POLR2A > TFRC > HPRT1 > ABL1 > GADD45A > HMBS > CDKN1A > RPL37A > UBC > GAPD H > CDKN1B > CASC3 > POP4 > PGK1 > GUSB > YWHAZ > PPIA > RPS17 > MRPL19 > B2M > EIF2B1 > ELF1 > PSMC4 > PUM1 > IPO8 (Fig. 1);

Summary

Introduction

Elimination of sources of error in the qRT-PCR technique, such as differences in the quantity and quality of extracted mRNA, the presence of contaminating genomic or operator DNA, divergences in reverse transcription and PCR efficiencies must occur for the qRT-PCR to be valid[16,17,18]. To ensure uniformity and reproducibility of published data, the “Minimum Information for publication of Quantitative real time PCR Experiments” (MIQE) guidelines suggest that the choice of and number of reference genes should be an essential part of all qRT-PCR studies Validation of this important step guarantees normalisation of resulting data[19]. We showed that a combination of 3 genes (IPO8, MRPL19 and PPIA) provided the best combination of normalisation factors in qRT-PCR studies of endometrial cancer and that geNorm qbase+226 is more robust than the other software packages in its statistical corrections for all the possible sources of experimental error listed above This conclusion remains applicable and supportable when comparing mRNA levels in type 1 endometrial cancer with type 2 endometrial cancer in the same gene expression study, recent studies have identified additional targeted biomarkers for individualised endometrial cancer. If both groups are under consideration, our previously published data remains the correct choice[4]

Objectives

Methods

Results

Conclusion