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Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma
Thangesweran Ayakannu1, Anthony H Taylor2, Jonathon M Willets1
1Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, UK.
Molecular Human Reproduction
|July 1, 2015
Summary
Selecting validated reference genes is crucial for accurate gene expression analysis in endometrial cancer. This study identified MRPL19, IPO8, and PPIA as the optimal combination for reliable qRT-PCR normalization in endometrial carcinoma tissues.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Real-time quantitative RT-PCR (qRT-PCR) is essential for gene expression analysis.
- Accurate normalization using stable reference genes is critical for reliable qRT-PCR data.
- No systematic evaluation of reference genes for endometrial cancer exists.
Purpose of the Study:
- To identify and evaluate suitable endogenous reference genes for normalization in endometrial cancer tissues.
- To assess the suitability of 32 potential reference genes using qRT-PCR.
- To establish a validated reference gene set for endometrial carcinoma studies.
Main Methods:
- Systematic evaluation of 32 candidate reference genes using TaqMan® array panels.
- Analysis of gene expression stability using geNorm, qBasePLUS, NormFinder, and BestKeeper software.
- Validation of selected reference genes by quantifying cannabinoid receptor 1 (CB1) mRNA expression.
Main Results:
- Multiple algorithms identified MRPL19, PPIA, and IPO8 as the most stable reference genes.
- geNorm identified PUM1, UBC, PGK1, MRPL19, and PPIA as a suitable combination.
- NormFinder suggested MRPL19 (single) and IPO8/PPIA (combination) as optimal.
- BestKeeper ranked MRPL19 as the most stably expressed gene.
- Validated reference genes (MRPL19, PPIA, IPO8) revealed significant CB1 mRNA differences between normal and malignant endometrium.
Conclusions:
- Arbitrary selection of reference genes in endometrial carcinoma qRT-PCR studies can lead to inaccurate results.
- MRPL19, IPO8, and PPIA represent a validated and reliable reference gene combination for endometrial carcinoma.
- Validated reference gene normalization is essential for robust gene expression profiling in cancer research.
