Validating internal controls for quantitative plant gene expression

Posted by / 27-Jan-2018 00:24

Validating internal controls for quantitative plant gene expression

The whisker caps represent the maximum and minimum values. Genes are in order from the most (lower Cq, on the left) to the least abundantly expressed (higher Cq, on the right).

Abbreviations: N: number of samples; CP: crossing point; GM [CP]: geometric CP mean; AM [CP]:arithmetic CP mean; Min [CP] and Max [CP]: CP threshold values; SD [±CP]: CP standard deviation; CV [%CP]: variance coefficient expressed as percentage of CP level; Min [x-fold] and Max [x-fold]: threshold expression levels expressed as absolute x-fold over- or under-regulation coefficient; SD [±x-fold]: standard deviation of absolute regulation coefficient. To obtain reliable results from q RT-PCR studies, two or more reference genes should be used for data normalization.

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.

Normalization requires the use of one or more reference genes (also called internal control genes) for which expression is constant and stable at different developmental stages, nutritional conditions or experimental conditions [].

Unfortunately, a gene has not been found for which expression is absolutely stable under all circumstances or across species that can be used indiscriminately for q RT-PCR analysis.

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 central horizontal line across the box is depicted as the median.

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.

Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene.

Its wood is known internationally for its beauty, weightlessness, durability and weather resistance and it is used in the building of ships, furniture, house floors and walls, and general carpentry [].

Despite the great economic importance of teak, there are no studies of gene expression, the genome sequence is not available and sequenced genes are limited.

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Vertical bars give a confidence interval for the inter-tissue variation.

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