Qpcrmix hs lowrox kit

Total RNA was isolated from cells or liver tissue using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), followed by precipitation with isopropanol, according to the manufacturer’s instructions. Liver tissue samples were homogenized in TRIzol using the Precellys^® homogenizer. A total of 0.5–1 μg of total RNA was further treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) and supplied with RiboLock RNase Inhibitor (40 U/μL) to the final concentration 0.4 U/μL. cDNA was generated using a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol. RNA levels were assessed by qPCR using qPCRmix-HS LowROX Kit (Evrogen, Moscow, Russia) in the Cycler CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The RNA levels of interest were normalized to the level of the mouse housekeeping genes GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (β-actin) and to the average value of the control group where needed. RT-qPCR was performed using specific primers listed in Table S3.

Total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. In order to remove any residual DNA from samples, isolated total RNA (0.5 μg) was further treated with 1U of DNase I (Thermo Fisher Scientific, Waltham, MA, USA), supplied with a RiboLock RNase Inhibitor (0.4 U/μL). cDNA was synthesized using a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), followed by qPCR using a qPCRmix-HS LowROX Kit (Evrogen, Moscow, Russia), according to the manufacturer’s protocols. qPCR was performed using primers listed in Supplementary Table S4. Positions of NRAS primers are shown in Supplementary Figure S2D. Gapdh was used as a reference gene for the RT-qPCR analysis.