Ellen De Keyser, Laurence Desmet, Jan De Riek
Currently in plant research, validated reliable RT-qPCR protocols are still rare. The last decade gene expression studies have been implemented widely in plant science. However, the methods used are often only semi-quantitative or quantification was not at all performed according to the MIQE-guidelines. The necessity of using multiple reference genes has become obvious in quite some cases, but assay-specific validation of these genes is often lacking. Still too often reference genes are selected for a species-wide application, no matter what treatment is given. Also RNA quality control is a crucial bottleneck. Machines for capillary electrophoresis allow to determine RNA quality quite easily, but how do you deal with this information? RIN or RQI values do not apply on plant material, since the training software was only developed using human/animal material. Plant material does not contain a 28SrRNA band but a 25S band. In addition, total RNA in chloroplast-containing plant tissues also consists of 16S and 23S rRNA adding 2 extra peaks. An alternative approach (visual evaluation) needs to be taken to decide on the quality of plant RNA samples. The use of noRT samples is another delicate point. Hardly any paper reports on the use of noRTs and in those cases noRTs were used after all, no information is available on the outcome. In our experience, the appearance of samples of which the Cq-value of the noRT is within 5 units of the actual sample are common in most experiments. Ignoring this information can lead to a severe overestimation of the gene expression in a specific sample. These three problems will be discussed more profoundly in view of the necessary application of the MIQE-guidelines in plant research.
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