5.1. Paenibacillus larvae gene expression
P. larvae gene expression under different experimental conditions can be investigated at the transcriptional level by making use of quantitative reverse transcriptase polymerase chain reactions (qRT-PCR). The state-of-the-art-analysis of qRT-PCR data relies on normalized and calibrated relative quantities (Vandesompele et al., 2002; Hellemans et al., 2007). In order to normalize the qRT-PCR data on target genes, normalization factors (NFs), based on the geometric mean of converted threshold cycle values (Ct-values), need to be calculated for each sample. Before calculating NFs, one has to decide how many reference genes should be included in this calculation. This decision is based on the expression stability of candidate reference genes under all examined experimental conditions. Therefore, the protocol below describes how to select reference genes for P. larvae.