, Six gestating females of each genotype were used at each analyzed stages. Individual RNA extractions were performed using the RNeasy Lipid Tissue Mini kit, RNA purifications and RT-(q)PCR
, Reverse transcription was performed on 5?g of total RNA, using Invitrogen SuperScript TMIII reverse transcriptase kit (18080051) or RTVilo (11754050) and random primers, according to the manufacturer's instructions. Amplification of the mouse Sprn cDNA ORF was performed using oligonucleotides 5?-taggcttgtgaccaattcttgcc-3? and 5?-ggagtttagcctggtctaaggc-3?, that are located on the two separate Sprn exons to avoid amplification of potential contaminating genomic DNA. PCR conditions were made of 40 amplifications cycles; 94 °C-30 s, 60 °C-60 s and 72 °C-30 s, using GC-rich adapted PCR amplification kit (KAPA2G Robust PCR kit, SigmaAldrich). Two WT and two Sprn 0/0 mouse brains were analyzed. For RT-qPCR, quantification was performed on triplicates, using 3 different RNA samples for each genotype, using the ABsolute Blue QPCR SYBR Green ROX Mix (Thermo Scientific) and standard PCR conditions. Primers were designed on separate exons, RNA concentration was calculated by electro-spectrophotometry and the RNA integrity checked with the Agilent Bioanalyser
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