Currently in plant research, validated reliable RT-qPCR protocols are still rare. The methods used are often 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. Also RNA quality control is a crucial bottleneck. Machines for capillary electrophoresis allow determining RNA quality quite easily, but RIN or RQI values do not apply on plant material. The use of noRT samples is another delicate point. In our experience, the appearance of samples of which the Cq-value of the noRT is within 5 units of the actual sample is 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 applicaton of the MIQE-guidelines in plant research.
|Title of host publication||Global Engage's qPCR and Digital PCR Congress|
|Number of pages||1|
|Place of Publication||Lyon|
|Publication status||Published - Sep-2013|
|Event||qPCR and Digital PCR Congress - Lyon, France|
Duration: 9-Sep-2013 → 10-Sep-2013