Real-Time PCR

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Is there a need for the reference gene(s) to be measured in the same plate (run) as the gene of interest?

In a relative quantification experiment, what you are most likely interested in is to compare the expression level of a particular gene among different samples. The most common way of correcting variations in the target nucleic acids input amount among samples is normalization by using reference genes. Assuming you have many samples and genes, you need to make a decision how to organize them in plates (runs).

There are in general two methods - sample maximization method and target maximization method (Hellemans et al., Genome Biology 2007, 8:R19). It is definitely recommended to use sample maximization whenever possible, that is use as many samples as possible for a given gene in the same run. This strategy does not suffer from run-to-run variation between samples (as all samples are measured in the same run for a particular gene), and therefore does not require inter-run calibration to be performed.

Reference genes must be taken as any other gene. Surprisingly perhaps, there is no need for the reference gene(s) to be measured in the same plate as the gene of interest. Even it is perfectly ok to normalize a gene of interest measured by a hydrolysis probe on qPCR instrument X with a reference gene measured by SYBR Green I on qPCR instrument Y. If you – like most of RT-qPCR users- are interested in relative quantification, then you only need to make sure that all samples are treated equally; it is ok however to measure genes differently.

There are data analysis tools that, unfortunately, do not support this strategy. For example, when analyzing your relative quantification experiment in Applied Biosystems’ software, you are forced to have your reference gene on the same plate like your target gene, otherwise the software is unable to calculate results. Not only in these cases, we definitely advice to use a tool compatible with these best practices in qPCR data analysis and we do offer the qbaseplus software, the gold standard and the leading qPCR software tool compatible with any qPCR instrument on the market to enable you do it.

 

Richard Nádvorník, richard.nadvornik@seqme.eu

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