Ribolock rnase inhibitor

Isolated RNA was spectrophotometrically quantified and afterwards, 250 ng of the total RNA was utilized for reverse transcription. As a first step, the remaining DNA was degraded by DNaseI (Thermo Fisher Scientific Inc., Waltham, USA). For the cDNA synthesis, oligo-(dT)₁₈-primers from the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc.) were used. The addition of 1 μL RiboLock RNase inhibitor (Roche) enhanced the quality of the cDNA.

Isolated mitochondria from WT and METTL8 KO cells were lysed in a buffer containing 3% sucrose, 100 mM KCl, 10 mM MgCl₂, 20 mM HEPES–KOH, pH 7.4, 1% digitonin, 1× Protease inhibitor cocktail (Roche) and 0.08 U/μl RiboLock RNase Inhibitor. Five hundred micrograms of mitochondrial extract were separated by centrifugation on 5–30% sucrose (w/v) gradients in 100 mM KCl, 10 mM MgCl₂, 20 mM HEPES/KOH, pH 7.4, 1× Protease inhibitor cocktail (Roche) at 79,000 × g, 4 °C for 15 h using an SW41 Ti rotor (Beckman Coulter)^{49 (link)}. Fractions (1–16) were collected with BioComp fractionator, and proteins and RNAs analysed by western blotting and northern blotting, respectively.