I'm running Real-time PCR on STEPONEplus instrument (singleplex) which allows me to choose only one endogenous gene. However, can I run another endogenous gene in the same 96 well? I would also want to know if I have to run these 2 endogenous gene every time I'm running Real Time or can I use 1 endogenous gene and the other in different sets of experiments but use the values from these sets to calculate the CT values?
Hi, the first question: Why do you want to use 18S and beta-actin? Cheers, Nadine
When doing relative quantification, there is no need that your references genes are measured in the same plate (run) as your genes of interest. It is perfectly okay to measure gene of interest in plate 1, and measure reference gene 1 in plate 2, and reference gene 2 in plate 3 (see Figure 2 - sample maximization strategy in attached paper).
Article Hellemans J, Mortier GR, De Paepe A, Speleman F, Vandesompel...
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