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Published on: February 21, 2014
Comparison of 11 endogenous control genes for normalization of mRNA obtained from paraffin-embedded tissues
Rekha Pai1, Prasanna Samuel, Arun G Nehru
1Department of Pathology, Christian Medical College, Vellore 632004, Tamil Nadu, India.
Abstract:
Real-time reverse transcriptase PCR (RT-PCR) based assays are being increasingly used in characterization of gene expression. Good quality mRNA is an essential prerequisite for such assays. While fresh tissues provide quality mRNA, the same may not be true of tissues which are formalin-fixed and paraffin-embedded (FFPE). This emphasizes the need to identify a good endogenous control gene to normalize for differences in quality and RNA recovery. We attempted to characterize gene expression patterns of 11 commonly used endogenous control genes among 20 FFPE tissues (both neoplastic and normal). Pearson's coefficient of correlation was determined by comparing the expression of each gene against the mean expression of all other genes. beta2 microglobulin (beta2M) and beta-actin (betaA) (r = 0.95 and 0.94, respectively) were found to be stably expressed across all tissues. However, betaA had greater accuracy (2 x SD) than beta2M and therefore may be a better choice of an endogenous control for experiments that require normalization while using FFPE tissues.
Insights
Formalin-fixed, paraffin-embedded tissues require reliable endogenous control genes for gene expression analysis. Beta-actin (betaA) demonstrated stable expression and superior accuracy compared to beta2 microglobulin (beta2M) in FFPE tissues.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Real-time reverse transcriptase PCR (RT-PCR) is crucial for gene expression analysis.
- High-quality mRNA is essential for accurate RT-PCR results.
- Formalin-fixed, paraffin-embedded (FFPE) tissues often yield compromised mRNA quality, necessitating normalization controls.
Purpose of the Study:
- To evaluate the stability and suitability of commonly used endogenous control genes for gene expression studies using FFPE tissues.
- To identify the most reliable endogenous control gene for normalizing RT-PCR data from FFPE samples.
Main Methods:
- Gene expression patterns of 11 endogenous control genes were analyzed in 20 FFPE tissue samples (neoplastic and normal).
- Pearson's coefficient of correlation was used to assess gene expression stability across samples.
- Accuracy was determined by calculating 2 x Standard Deviation (SD) for candidate genes.
Main Results:
- Beta-2 microglobulin (beta2M) and beta-actin (betaA) exhibited stable expression across all tested FFPE tissues (r = 0.95 and 0.94, respectively).
- Beta-actin (betaA) demonstrated higher accuracy (lower 2 x SD) than beta2M.
- Both beta2M and betaA are suitable endogenous controls, but betaA offers improved normalization precision.
Conclusions:
- Beta-actin (betaA) is a highly reliable endogenous control gene for gene expression normalization in FFPE tissues.
- Selection of an appropriate endogenous control is critical for accurate gene expression profiling from FFPE samples.
- This study provides valuable guidance for researchers utilizing FFPE tissues in gene expression studies.
