Brilliant 2 sybr low rox master mix

Total RNA was isolated using a Qiagen RNeasy Mini Kit (Qiagen) and genomic DNA was removed using TurboDNAse (Ambion), and DNase removed using DNAse Inactivation Reagent (Ambion) according to the manufacturer's instructions. Synthesis of cDNA was carried out with 100 ng RNA per sample using an Omniscript RT kit (Qiagen). qPCR reactions were performed using Brilliant II SYBR low ROX master mix (Agilent) with relevant primers (Supporting Information Table S6) using an Applied Biosystems 7500 real time PCR machine. qPCR reactions were performed with 1 µl cDNA diluted ten‐fold with the exception of the mRNA decay assays where the cDNA was diluted 100‐fold. Relative quantification of mRNA was performed with each sample normalised to levels of actin transcript. Unless stated otherwise, qPCR data are presented as arbitrary units (2^‐ΔCt). Primers used for qPCR to detect VSG221 transcript amplify a region of the VSG221 gene located at 1058–1184 bp, and downstream of the region used for VSG221 RNAi (106–910 bp). Predicted RNA secondary structure was determined using RNAfold (University of Vienna) (ViennaRNA Package 2.0) (Gruber et al., 2008).

Total RNA was extracted using a Qiagen RNeasy Mini kit with genomic DNA depleted using Turbo DNase (Ambion) according to manufacturer's instructions. Reverse transcription with 100 ng of total RNA was carried out using a Qiagen Omniscript RT kit with random hexamers from Promega. Generated cDNA was diluted 10-fold and the equivalent of 0.5 ng of RNA was used as the template for the qPCR reactions. qPCR reactions were carried out using an Applied Biosystems 7500 real time PCR machine using Brilliant II SYBR low ROX master mix (Agilent) with primers listed in Supplementary Table S4.