Quantifying CLEC16A Expression by qPCR

As described in our previous publication^{37 (link)}, total RNA was isolated with Trizol reagent (Invitrogen) following RNA purification using the RNeasy Mini Kit (Qiagen) and converted to cDNA by High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer’s protocols. Assay comprising known human and murine CLEC16A RNA transcripts as well as control genes (β-actin and HRPT1) were measured by real time PCR on a ViiA 7 Real Time PCR System using predesigned 20X FAM-MGB TaqMan gene expression assays available from Applied Biosystems. All assays had primers covering exon-exon boarders to avoid DNA contamination. Triplicates were used for all samples included in the experiment. All PCR runs were performed on ViiA 7 Real Time PCR System using ViiA7 RUO software v1.2.2 (Life Technologies).