Division I.4.5 Pooling of samples for RT-qPCR

As the RT-qPCR protocols given are of similar or higher sensitivity than the cell cultivation methods, it may be acceptable to use supernatant from homogenised fish tissue material of pooled organs from up to 10 fish in cell culture medium for PCR. However, due to the much smaller inoculum used for PCR compared to cell cultivation, all fish tissues shall be carefully homogenised before collating material for extraction.

The same principle shall also be applied if samples are collected in RNA stabilization reagents. However, in that case it is often difficult to collect representative material from up to 10 fish in one tube, and the number of fish per pool shall therefore be reduced to 2 to 5.