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Identification of novel reference genes based on MeSH categories
Tulin Ersahin1, Levent Carkacioglu2, Tolga Can2
1Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey.
Plos One
|April 1, 2014
Summary
Identifying stable reference genes is crucial for accurate gene expression analysis. This study found that tissue-specific reference gene sets, particularly ribosomal genes, offer more reliable normalization than commonly used housekeeping genes across diverse experiments.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Accurate mRNA expression analysis relies on stable reference genes for normalizing transcriptome data.
- Gene expression variability necessitates comparable datasets across different experiments and laboratories.
- Existing housekeeping genes often exhibit tissue-specific expression, impacting data reliability.
Purpose of the Study:
- To identify novel, constitutively and stably expressed reference gene sets specific to different tissue origins.
- To evaluate the variability of gene expression across diverse microarray datasets.
- To establish context-dependent reference gene lists for accurate normalization in large-scale gene expression studies.
Main Methods:
- Analysis of 9090 microarray samples from 381 NCBI Gene Expression Omnibus (GEO) datasets.
- Utilized randomization and Receiver Operating Characteristic (ROC) curves to identify candidate reference genes.
- Calculated coefficient of variation and percentage of occurrence for gene stability assessment.
- Classified gene sets using Medical Subject Headings (MeSH) for tissue specificity.
- Validated tissue-specific reference gene candidates using RT-qPCR in carcinoma cell lines.
Main Results:
- Cell type-specific reference gene sets showed lower variability compared to a universal set.
- Identified novel, origin-specific reference gene sets based on expression stability and occurrence.
- Commonly used housekeeping genes (GAPDH, Actin, EEF2) exhibited significant tissue-specific variations.
- Several ribosomal genes were identified as highly stable reference genes in vitro.
- RT-qPCR validation confirmed the tissue-specific expression patterns of candidate reference genes.
Conclusions:
- Context-dependent reference gene sets are essential for reliable normalization of gene expression data.
- Tissue-specific reference genes, especially ribosomal genes, provide more accurate normalization than traditional housekeeping genes.
- Using two or more validated reference genes in combination is recommended for large-scale gene expression analyses (microarray, next-generation sequencing).
- The study provides comprehensive lists of novel reference gene sets classified by tissue type (MeSH).
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