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microRNAs as reference genes for quantitative PCR in cotton
Anna Karoline Silva Fausto1, Tatiane da Franca Silva1,2, Elisson Romanel1,2
1Lab. Virologia Molecular Vegetal, Depto. Virologia, IMPPG, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brasil.
Plos One
|April 18, 2017
Summary
Cotton microRNAs (miRNAs) are validated as reliable reference genes for gene expression normalization. This study demonstrates their stability across different tissues and under biotic stress, improving molecular analyses in cotton.
Area of Science:
- Plant Molecular Biology
- Genomics
- Biotechnology
Background:
- Accurate gene expression analysis in cotton (Gossypium hirsutum) is crucial for understanding fiber quality and plant fitness.
- Reliable reference genes are essential for normalizing quantitative real-time reverse transcription (RT)-PCR data.
- Existing mRNA reference genes in cotton have limited reported suitability for normalization.
Purpose of the Study:
- To identify and validate microRNAs (miRNAs) as stable and reliable reference genes for gene expression normalization in cotton.
- To assess miRNA stability across various tissues and under biotic stress conditions.
Main Methods:
- Assessed the stability of cotton miRNAs and known mRNA reference genes in root, stem, leaf, and flower samples from three cultivars.
- Utilized geNorm, NormFinder, BestKeeper, and ΔCt algorithms for reference gene selection.
- Validated selected reference genes under biotic stress (Cotton leafroll dwarf virus infection) and in specific tissues.
Main Results:
- MicroRNAs (miRNAs), specifically miR172, miR168, and miR390, were identified as the most stable reference genes in 8 out of 12 tested sets.
- Validated reference genes demonstrated excellent performance in normalizing gene expression under biotic stress and in specific tissues.
- miRNAs proved to be superior reference genes compared to traditional mRNA candidates.
Conclusions:
- MicroRNAs (miRNAs) are highly reliable reference genes for normalizing gene expression in cotton (Gossypium hirsutum).
- This finding supports the use of miRNAs for accurate gene expression studies in both miRNA and protein-coding genes in cotton.
- The validated miRNAs offer improved molecular analysis tools for cotton research.