Pyromark onestep rt pcr kit

Pyrosequencing validations were performed in cerebella derived from a different batch of F1 hybrids from that used in the RNA-seq experiments. The same age of animals was used for the pyrosequencing validations whenever the RNA-seq data showed an age-specific parental bias. Otherwise, either age group was used. RNA purification and quality control followed the same procedures described for the RNA-seq data. An average of three SNPs suitable for pyrosequencing analyses were identified for each candidate gene. Previously described three-primer strategy (Royo et al., 2007 (link)), including a 3′-biotinylated primer common to all reactions, was employed for the amplification of each targeted SNP. All primers were designed using Pyromark Assay Design 2.0. Pyromark One Step RT-PCR kit (Qiagen) was used for the amplification of each targeted region, followed by purification of single stranded biotinylated DNA according to the manufacturer instructions. Pyrosequencing was performed on the Pyromark Q96 MD (Qiagen). For each SNP at least 12 replicates were separately analyzed. Statistical analyses of the pyrosequencing data for each SNP were performed using BRAIM.