Validating internal controls for quantitative plant gene expression studies

To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet.

The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop.

Reverse transcription-q PCR (RT-q PCR) has become a popular method for gene expression studies.

Its results require data normalization by housekeeping genes.

No single gene is proved to be stably expressed under all experimental conditions.

Therefore, systematic evaluation of reference genes is necessary.

Data normalization based on reference genes is essential for obtaining reliable results for q RT-PCR assays.

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Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a Pg AP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions.

With the aim to identify optimum reference genes for RT-q PCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β), elongation factor 1-gamma (EF1-γ), eukaryotic translation initiation factor 3G1 (IF3G1), eukaryotic translation initiation factor 3B (IF3B), actin (ACT), actin11 (ACT11), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and cyclophilin ABH-like protein (CYC), using four widely used computational programs: ge Norm, Normfinder, Best Keeper, and the comparative ΔCt method.

The results were then integrated using the web-based tool Ref Finder.

Pair-wise analysis showed that two reference genes were sufficient for proper normalization, except when analyzing the gene expression studies in all samples set.

Results of this study can help in the selection of reference genes for quantitative real time PCR (q RT-PCR) normalization in pearl millet that will contribute towards more accurate and reliable quantification of transcripts in this important crop of the drylands.

were selected based on the available literature, and their expression stabilities were studied to determine their suitability for normalizing gene expression in pearl millet.

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