Absolute blue qpcr mix with sybr green and rox

Cells were harvested by centrifugation and snap frozen in liquid nitrogen. RNA was extracted using the RNeasy kit (QIAGEN) via mechanical disruption. 300 ng of total extracted RNA was used for reverse transcription. The RNA was first treated with DNase I using the DNA-free kit (Ambion) according to the protocol of the manufacturer. Reverse transcription was performed according to the protocol of the manufacturer using Superscript II reverse transcriptase (Invitrogen) with random hexamer primers. Quantitative real-time PCR was performed on a StepOnePlus Instrument (Invitrogen) using Absolute Blue QPCR Mix with SYBR Green and ROX (Thermo Fisher Scientific). All experiments were performed in three technical replicates and three biological replicates. Data were analyzed by the comparative cycle threshold method using ACT1 as endogenous control.

qPCR was performed as described previously (Dultz et al., 2016 (link)): cells at OD₆₀₀ 0.8–1 (1 ml) were harvested by centrifugation and snap-frozen in liquid nitrogen. RNA was extracted using the RNeasy kit from Qiagen via mechanical disruption. Total extracted RNA (300 mg) was used for reverse transcription. The RNA was first treated with DNase I using the DNA-free kit from Ambion according to the protocol of the manufacturer. Reverse transcription was performed according to the protocol of the manufacturer using Superscript II reverse transcriptase (Invitrogen) with random hexamer primers. qPCR was performed on a StepOnePlus Instrument (Invitrogen) using Absolute Blue QPCR Mix with SYBR Green and ROX (ThermoFisher Scientific). All experiments were carried out in three technical replicates and three biological replicates. Data were analyzed by the comparative CT method using ACT1 as endogenous control. Primers used for qPCR are listed in Supplemental Table 2.