Selection of reliable reference genes in eutopic and ectopic endometrium for quantitative expression studies.

Area of Science:

• Gynecology
• Molecular Biology
• Genetics

Background:

• Menstrual cycle phases influence endometrial and endometriosis tissue gene expression.
• Housekeeping genes, often used as reference genes (RGs), may exhibit variable expression.
• Accurate normalization in quantitative gene expression studies relies on stable RGs.

Purpose of the Study:

• To evaluate the stability of seven potential reference genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT, PPIA) in eutopic and ectopic endometrial tissues.
• To identify the most reliable RGs for normalization in reverse transcription quantitative polymerase chain reaction (RT-qPCR) studies of endometriosis.
• To provide recommendations for RG selection in endometriosis research.

Main Methods:

• Gene expression analysis using RT-qPCR in endometrial tissue samples from women of reproductive age.
• Assessment of RG stability using geNorm, NormFinder, and BestKeeper algorithms.
• Statistical analysis of gene expression data using Statistica software.

Main Results:

• Reference gene expression levels varied, with ACTB and B2M showing high expression, GAPD medium, and G6PD/HPRT low.
• RG stability was not uniform across all tested genes.
• PPIA, GAPD, and B2M were identified as the most stable RGs, while GUSB and G6PD were the least stable.

Conclusions:

• A minimum of two reference genes should be utilized for RT-qPCR analysis in endometriosis.
• PPIA and GAPD are recommended as the most suitable reference genes for gene expression studies in eutopic and ectopic endometrium.