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Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries

  • Reproductive Physiology and Disease
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes.

Methods

RNA was isolated from 5 normal human ovaries (52–79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49–75 years of age), and 4 PCOS ovaries (18–35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate.

Results

Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes.

Conclusions

This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

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Acknowledgments

We are grateful to Mira Hryniuk for reviewing the English language of the manuscript and Dolores Gonzalez and Olivier Van Kerk for their technical assistance. We are also thankful to Professor Etienne Marbaix for his kind collaboration and providing ovarian control and cancerous samples.

Funding

This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C. A. Amorim is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C. A. Amorim; grant 5/4/150/5 awarded to M. M. Dolmans; grant ASP-RE314 awarded to P. Asiabi; FNRS-PDR Convention T.0077.14 and EOS grant 30443682).

Author information

Authors and Affiliations

  1. Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200, Brussels, Belgium

    P. Asiabi, M. C. Chiti, M. M. Dolmans & C. A. Amorim

  2. Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, 1200, Brussels, Belgium

    J. Ambroise & B. Bearzatto

  3. Department of Clinical and Experimental Biomedical Sciences, University of Florence, 50134, Florence, Italy

    C. Giachini & M. E. Coccia

  4. Gynecology Department, Cliniques Universitaires Saint-Luc, 1200, Brussels, Belgium

    M. M. Dolmans

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  1. P. Asiabi
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Contributions

P. A.: study design, experimental procedures, analysis, interpretation of data, and manuscript preparation. J. A.: statistical analysis. C. G. and M. E. C.: PCOS tissue supply. B. B.: manuscript revision. M. C. C.: PCOS tissue supply and manuscript revision. M. M. D.: manuscript preparation and revision. C. A. A.: experimental design, experimental procedures, interpretation of results, and manuscript revision.

Corresponding author

Correspondence to C. A. Amorim.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Use of human ovarian tissue was approved by the Institutional Review Board of the Université Catholique de Louvain on November 28, 2016 (IRB reference 2012/23MAR/125, registration number B403201213872).

Four PCOS samples came from the assisted reproduction technology center of the Careggi University Hospital in Florence, Italy (Ethical approval n. 11314_bio).

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Supplementary Figure 1

Standard deviation for qPCR technical triplicates as a function of the average value of qPCR technical triplicates. A sharp increase in variability was observed for Ct > 35. (PNG 752 kb)

High resolution image (TIF 146 kb)

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Asiabi, P., Ambroise, J., Giachini, C. et al. Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries. J Assist Reprod Genet 37, 2545–2553 (2020). https://doi.org/10.1007/s10815-020-01901-8

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  • DOI: https://doi.org/10.1007/s10815-020-01901-8

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