Gotaq real time qpcr master mix

1 μg of total RNA was diluted to a final volume of 11 μl. 2 μl of random primers (Promega) were added after which the mixture was incubated at 65 °C for 5 min. A master mix containing Transcriptor Reverse Transcriptase (Roche), Reverse Transcriptase buffer, 2 mM dNTP mix and RNasin Ribonuclease Inhibitors (Promega). This mixture was incubated at 25 °C for 10 min, then 42 °C for 40 min and finally 70 °C for 10 min. The resulting cDNA was then diluted 1:2.5 in H₂O for subsequent use. qPCR was performed using a Step-One Plus Real-Time PCR System (Thermofisher Scientific). Either Taqman (ThermoFisher Scientific) probes with GoTaq Real Time qPCR Master Mix (Promega) or primers (Sigma) with PowerUp SYBR Green Master Mix (ThermoFisher Scientific) were used. All probe and primer details can be found in Supplementary Data 5 and 6. The enrichment was normalised with control mRNA levels of GAPDH and relative mRNA levels were calculated using the ΔΔCt method comparing to control group.

Total RNA was diluted to a final volume of 11 µl. Two microliters of random primers (Promega) were added after which the mixture was incubated at 65 °C for 5 min. A master mix containing Transcriptor Reverse Transcriptase (Roche), Reverse Transcriptase buffer, 2 mM dNTP mix and RNasin Ribonuclease Inhibitors (Promega) was then added. This mixture was incubated at 25 °C for 10 min, then 42 °C for 40 min and finally 70 °C for 10 min. The resulting cDNA was then diluted 1:2.5 in H₂O for subsequent use. qPCR was performed using a Step-One Plus Real-Time PCR System (Thermofisher Scientific). Taqman (ThermoFisher Scientific) probes with GoTaq Real Time qPCR Master Mix (Promega) were used. The enrichment was normalised with control mRNA levels of GAPDH and relative mRNA levels were calculated using the ΔΔCt method compared to the control group. For the list of probes see Supplementary Data 2.