Validating internal controls for quantitative plant gene expression
Their stability as reference genes was validated by Norm Finder, Best Keeper, ge Norm and Delta Ct programs.Results obtained from all programs showed that This study proposes a first broad collection of teak tissue and organ m RNA expression data for nine selected candidate q RT-PCR reference genes.One or more appropriate reference genes are crucial to accurately compare m RNA transcripts through different tissues/organs and experimental conditions.Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak.L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world.Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (q RT-PCR) is a sensitive technique employed for quantifying gene expression levels.
However, some variables such as the integrity, amount and purity of the RNA used as well as enzyme efficiency during c DNA synthesis and PCR amplification make an additional step to normalize the data necessary .
To compare the differences in transcript levels between reference genes, the Cq range was determined and the coefficient of variance was calculated for each gene across all samples based on the interquartile range (25-75% percentiles).
The average Cq values of the different genes ranged from 22 to 34 cycles (Figure samples.
The annealing temperature of 65°C was effective for all primers; nevertheless, its choice can impact on the efficiency of the reaction.
Altogether, the results showed that the chosen primers accurately amplified the candidate reference genes.
To date, q RT-PCR reference genes have not been identified and validated for teak. Expression profiles of these genes were evaluated by q RT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak.