Quantifying rRNA Methylation Levels

The levels of rRNA methylation were analyzed by RT-QPCR^{68 (link)}. A reverse-transcription primer R and a pair of primers (F1 and R1) near A674 on 26S rRNA were designed for QPCR. The sequences of the primers were R: 5′-AGTCACAAGTGACACGCAC-3′; F1: 5′-ACAGTGTTGCCCATCTCGC-3′; R1: 5′-ACGTCGGCCAATTCGAGAC-3′. The 26S rRNA transcript was specifically reversed transcribed using primer R to generate cDNA with 100 ng of total RNA extracted from either WT or rrp-8(kun54) mutant worms and either 10 μM or 1 mM dNTPs in each sample. The reverse transcription system included 200U Hscript Reverse Transcriptase (Vazyme Biotech), 40U RNase inhibitor (Vazyme Biotech), 1 μM specific reverse primer R, and either 10 μM and 1 mM dNTP in each sample. For each reaction, reverse transcription was performed at 25 ℃ for 5 min, followed by an incubation at 50 ℃ for 15 min and at 85 ℃ for 5 min. RT-QPCR was performed to quantify the specific cDNA levels using the primer pair F1/R1 spanning the methylation site. The relative methylation rate of a specific site on 26S rRNA was calculated as the ratio of the cDNA levels obtained using 1 mM dNTPs to that obtained using 10 μM dNTPs in each sample.