Brilliant 2 sybr green qpcr low rox master mix

RNA transcript analysis was performed using quantitative reverse transcription PCR (qRT-PCR). Total RNA was isolated using the RNeasy kit (Qiagen) and DNase treated with TURBO DNA-free kit (Invitrogen). Reverse transcription was performed using 100 ng RNA as a template for cDNA synthesis using random hexamer primers (Promega) and the Omniscript RT kit (Qiagen). qPCR was performed on the 7500 Fast Real-Time PCR system (Life Technologies) using Brilliant II SYBR Green QPCR Low ROX Master Mix (Agilent Technologies). DNase treated RNA without reverse transcriptase was used as a control. The amplification conditions for each primer pair were optimised, and the primer sequences are listed in S1 Table. Transcript levels were normalised against actin mRNA, and plotted as relative change relative to the zero hour time point.

mRNA expression levels were analysed using quantitative reverse transcriptase PCR (qRT-PCR). Total RNA was isolated from T. brucei cells under RNAse-free conditions using RNeasy kits (Qiagen; 74104), followed by DNase treatment with the TURBO DNA-free kit (Invitrogen; AM1907). Reverse transcription of cDNA was performed with the Omniscript RT kit (Qiagen; 205111) using 100 ng of template RNA and 5 mM random hexamers (Promega; c1181) according to the manufacturer’s instructions. cDNA was diluted tenfold before use in qPCR reactions with Brilliant II SYBR Green qPCR Low ROX Master Mix (Agilent technologies; 600830). qPCR reactions were performed with a 7500 Fast Real-Time PCR system (Life Technologies).
For Morpholino transfection experiments, time points for genes of interest were normalised against the untreated sample and plotted as a relative change to the initial time point. Transcript quantitation after SLAP1 RNAi was performed after normalisation against actin for the respective time point. All primers used for qPCR are in Supplementary Table 2.

qPCR was performed in triplicate per donor for each group within the same qPCR-run. A calibration curve was run simultaneously on the RG candidate tested on the donors. Only data obtained from runs fulfilling the same criteria for efficiency and correlation coefficient as the primer verification was included for analysis. The Cq threshold was set to the value of 0.1 for all qPCR runs. Brilliant II SYBR®green QPCR Low ROX master mix (Agilent, cat.no. 600806) was used with a total reaction volume of 25 μl in 96-well optical reaction plates (Agilent, cat.no. 401333) with 5 μl of diluted cDNA. The plate was sealed with optical plastic caps (Agilent, cat.no. 401425). qPCR was performed using Mx3000 (Stratagene) and the results were collected using Mx3000 version 4.0 software for Windows (Stratagene). The reaction was initiated by heating to 95°C for 10 min., followed by 40 cycles elongation at 60°C for 1 min. and denaturation at 95°C for 30 sec.
To verify the chosen RGs, a normalization experiment was set up. vWF was used as target gene, and normalized to different combinations of RG candidates. The level and the standard deviations of the fold changes between VEGF treated cells and controls were compared for normalization to different RG combinations. The fold changes in vWF expression between VEGF treatment and controls were calculated with the 2^-∆∆Cq-method.