Quantifying Signaling Effects of Mutations

To validate the signaling effect triggered by the presence of mutations, we measured the mRNA expression of downstream target genes by qRT-PCR. Total RNA (1 μg) was treated with DNAse and reverse-transcribed into cDNA with 50-μM oligo(dT)20 using a Superscript III transcriptase kit (Thermo Fisher Scientific). About 2 μL of cDNA was used in a 12-μL PCR reaction containing 1× SYBR Green PCR Master Mix (Applied Biosystems) and 3 pmol of each specific primer for the target gene. As a reference gene, we used ribosomal protein S8 (RPS8). The reaction was performed in triplicate on a QuantStudioTM 12K Flex (Applied Biosystems). The relative expression levels were calculated based on Delta-Delta-CT (ddCT), as previously reported [42 (link),43 (link)]. The Kruskal-Wallis test was applied to analyze our set of candidates.