RNA isolation, cDNA synthesis and quality control were performed as described earlier52. mRNA expression was examined by an optimized three-step real-time quantitative PCR assay following the protocol described before53. Besides ADNP itself, ADNP2 was included based on the reported correlation of expression in human brain tissue31. TMPO, CCNC and PLAGL2 were reported to be significantly downregulated in homozygous Adnp knockout mice embryos, while ABCF3 was reported to be upregulated in heterozygous Adnp knockout mice embryos18,23. Finally, TP53 is upregulated in HT29 cells incubated with ADNP antioligodeoxynucleotide32. YWHAZ and HPRT were selected as reference genes, according to geNorm calculations54. qPCR primers were selected from literature31,55, the RTPrimerDB56 or designed using an in-house automated pipeline (see URLs), conforming to requirements of intron-spanning location, no SNP content, no dimer formation at the 3’ end of the primers, and low amplicon folding, with no folding in primer binding sites. The amplification efficiency of the different primers was assessed and confirmed to be above 1.85. Primer sequences are available on request. Expression values of two cDNA syntheses originating from two different RNA isolations per patient
