Selection of suitable reference genes for gene expression studies in normal human ovarian tissues, borderline ovarian tumours and ovarian cancer

Abstract

The use of reference genes is the most common method of controlling the variation in mRNA expression during quantitative polymerase chain reaction, although the use of traditional reference genes, such as β‑actin, glyceraldehyde‑3‑phosphate dehydrogenase or 18S ribosomal RNA, without validation occasionally leads to unreliable results. Therefore, the present study aimed to evaluate a set of five commonly used reference genes to determine the most suitable for gene expression studies in normal ovarian tissues, borderline ovarian and ovarian cancer tissues. The expression stabilities of these genes were ranked using two gene stability algorithms, geNorm and NormFinder. Using geNorm, the two best reference genes in ovarian cancer were β‑glucuronidase and β‑actin. Hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase were the most stable in ovarian borderline tumours, and hypoxanthine phosphoribosyltransferase‑1 and glyceraldehyde‑3‑phosphate dehydrogenase were the most stable in normal ovarian tissues. NormFinder ranked β‑actin the most stable in ovarian cancer, and the best combination of two genes was β‑glucuronidase and β‑actin. In borderline tumours, hypoxanthine phosphoribosyltransferase‑1 was identified as the most stable, and the best combination was hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase. In normal ovarian tissues, β‑glucuronidase was recommended as the optimum reference gene, and the most optimum pair of reference genes was hypoxanthine phosphoribosyltransferase‑1 and β‑actin. To the best of our knowledge, this is the first study to investigate the selection of a set of reference genes for normalisation in quantitative polymerase chain reactions in different ovarian tissues, and therefore it is recommended that β‑glucuronidase, β‑actin and hypoxanthine phosphoribosyltransferase‑1 are the most suitable reference genes for such analyses.

Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

“…Five commonly used reference genes were selected: actin beta (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and TATA-box binding protein (TBP) (Santin et al, 2013;Weber et al, 2014;Iser et al, 2015;Ofinran et al, 2016;Wang et al, 2016a). RT-qPCR was performed using StepOnePlus Real-Time PCR cycler.…”

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

“…The widely used ribosomal RNA (18S or 28S rRNA), for example, is not the most appropriate gene mainly because its expression is much higher and its degradation inferior to those of the target mRNA, the rRNA:mRNA ratio is highly variable between different samples, and its transcription pathway is independent of that of mRNA [21][22][23]. Furthermore, the selection of a reference gene based only on a literature search is equally inadequate, because the expression levels of several constitutive genes, such as GAPDH and ACTB, is known to vary considerably in the same tissue when tested under different experimental conditions [24][25][26]. Thus, previous reference gene validation is essential for each tissue and study, based on a set of candidate genes.…”

<p>Identification and Validation of Reference Genes Selection in Ovarian Cancer Exposed to Hypoxia</p>

“…For example, GAPDH and βactin were always selected as potential candidate reference genes, based on previous experience. However, previous studies have shown that the expression levels of these housekeeping genes (HKGs) can vary between cell types [6][7][8] and in different experimental conditions. 9,10 Therefore, prior to their initial use, the stability of the expression of certain HKGs should be validated first.…”