Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

Olsvik, Pål A; Lie, Kai K; Jordal, Ann-Elise O; Nilsen, Tom O; Hordvik, Ivar

same time, the A260/230 ratio dropped from 2.4 to 2.1. The DNase treatment therefore adds substances to the RNA solution that increases the absorbance at 230 nm more than it decrease the 260 nm absorbance. The added substance (salt or some other component) may inhibit the RT reaction or the PCR reaction, sometimes called PCR poisoning. We have seen that the A260/230 ratios are quite low in samples that give inadequate PCR efficiency slopes, especially with RNA from head kidney, thymus and intestine tissues, in which the gradient of the standard curve is less than -3.3 ( Table 2 ). The reason one obtain better amplification rate efficiencies with the more diluted samples is because the inhibitor has been diluted below its effective level. The obvious way around this problem is to dilute the amount of cDNA put into the PCR reaction. Alternatively, cleanup columns can be used to purify and concentrate the RNA.