We applied the previously described stepwise approach on our data set consisting of 46 RNA families. We shuffled for each family 1000 times with a conservative method which mononucleotidely shuffles only columns with the same pattern of gaps and conservation. Then we averaged over the significance values of all families. Using a significance level θ = 0.01, we got a threshold for high reliable base pairs of p threshold However, the parameter tuning has indicated that the performance of reliability thresholds depend on another parameter (the weighting factor for single-stranded positions α) which has high impact in the RNA structure prediction of PETfold, and that slightly different reliability thresholds perform better for the data set.