The RTL-P approach for detecting the presence of 2'-O-methylation in RNA. (A) A schematic of the primer-design strategy for RTL-P. The orientations of the primers are indicated by the arrows. (B and C) A schematic illustration of the RTL-P procedure used to detect the presence (B) or absence (C) of 2'-O-methylated sites in RNA. (1) RNA preparation by DNase I treatment to remove the DNA contaminants. (2) RT with a RT primer at a low or high dNTP concentration. At a low dNTP concentration, the RT reaction is impeded by the 2'-O-methyl groups, resulting in shorter RT products. At a high dNTP concentration, the RT reaction is not impeded and therefore does not produce shorter cDNA products. (3) PCR with different PCR primer pairs to amplify the RT products. The PCR reaction with the R/F_U primer pair only amplifies the longer cDNA products, but amplification with the R/F_D primer pair generates both the longer and shorter cDNA products. For RNAs with methylation sites, the quantity of the R/F_U PCR product is less than the R/F_D PCR product when the RT products generated at a low dNTP concentration are used as the amplification template. However, the quantity of the PCR product generated by the R/F_U primers is equal to the R/F_D PCR product when the RT products generated at a high dNTP concentration are used as the template. (4) Gel electrophoresis of the RT-PCR products. If the 2'-O-methylated site is present in the RNA analyzed, the R/F_U band is weaker than the R/F_D band in the low dNTP lane. In contrast, the R/F_U band is nearly equal to or stronger than the R/F_D band in the high dNTP lane. The intensity difference depends on the length of the PCR products generated from the R/F_U primers versus the R/F_D primers. m = 2'-O-methylation.

The detection of 2′-O-methylation in human rRNAs by RTL-P. (A) A schematic diagram of the primer design for RTL-P to detect a region enriched with 2'-O-methylated nucleotides (Cm2338, Am2350, Cm2352, Am2388, Um2402, Cm2409, Gm2411) in the human 28 S rRNA. (B) The detection of several 2'-O-methylated sites in human 28 S rRNA by RTL-P at different dNTP concentrations. Twelve PCR cycles were used for the amplification. Approximately 200 ng of total RNA was used in each RT reaction. (C) A schematic of the primer-design for RTL-P to detect the Gm1490 methylation in the human 18 S rRNA. (D) The detection of Gm1490 by RTL-P under different template concentrations, dNTP concentrations and PCR cycles. (E) Correlation of signal intensity ratio of amplification products to the PCR cycle number for RTL-P assay at low dNTP concentrations. The ratios of PCR signal intensity are indicated beneath each lane. A linear range of signal ratios versus cycle number is shown in the right panel. All assays were independently performed at least three times with the representative results of a single experiment shown in the figure.

The RTL-P strategy for the detection of the exact location of a 2'-O-methylated site in RNA. (A) A schematic diagram of the primer design for the RT reaction and the PCR. m = 2'-O-methylation. (B) A schematic representation of the RTL-P procedure used to detect the exact location of the 2'-O-methylation. (1) RNA preparation by DNase I treatment to remove the DNA contaminants. (2) RT with the MeUA-RT or MeA-RT primers in different reaction tubes (①–④) at low or high dNTP concentrations, respectively. At a low dNTP concentration, the RT reaction with the UA-RT primer (①) is impeded by the 2'-O-methyl group and produces less cDNA products than that the MeA-RT primer (②). At a high dNTP concentration, the cDNA products generated with MeUA-RT primer (③) is equal to the MeA-RT primer (④). (3) PCR with the same PCR primer pair P_F/P_R to amplify the RT products. The quantity of PCR product generated from the MeUA-RT cDNA (①) is less than the quantity from the MeA-RT cDNA (②) when the residue of interest is 2'-O-methylated. In contrast, the quantity of PCR product generated from the MeUA-RT (③) and MeA-RT cDNAs (④) are equal when the residue analyzed is unmethylated. (4) Gel electrophoresis of the RT-PCR products. If the analyzed nucleotide is 2'-O-methylated, the band of the RT-PCR products generated from the MeUA-RT cDNA (①) is weaker than the band of the products generated from the MeA-RT cDNA (②) when a low concentration of dNTPs is used in the RT; however, the bands of the RT-PCR products generated from the MeUA-RT cDNA (③) and the MeA-RT cDNA (④) are equal when a high concentration of dNTPs is used. If the analyzed nucleotide is unmodified, no differences in band intensities of the RT-PCR products are observed regardless of the concentration of dNTPs used. N = any nucleotide base.

The detection of the Am1164 2'-O-methylation in the 25S rRNA of S. pombe by RTL-P. (A) The potential base-pairing interaction between the snR61 snoRNA and its target region in the 25S rRNA of S. pombe. (B) A schematic diagram of the RT primer design. (C) An analysis of Am1164 by the traditional primer extension method in wild type (Wt) and mutant (△snR61) S. pombe. The primer extension was performed in the presence of increasing dNTP concentrations (4 μM and 0.15 mM). A subset of the rRNA sequence is indicated on the left. The position of the reverse transcriptase stop site is indicated by an arrow, and the methylated site is marked with an asterisk. T/G/C/A = the rDNA sequence ladders. (D and E) The detection of Am1164 by RTL-P at varying dNTP concentrations with 20 ng (D) and 2 ng total RNA (E) in each RT reaction. All assays were independently carried out at least three times, and the representative results of a single experiment are shown. The ratio of PCR signal intensity is shown beneath each lane.

The detection of 2'-O-methylation at the 3'-ends of small RNAs. (A) An experimental scheme for the detection of 2′-O-methylation by RTL-P. (B) The detection of 2'-O-methylation at the 3'-ends of piRNAs and miRNAs. The 3' modification of small RNAs (piRNAs) is identified by the difference in the signal intensities of the RT-PCR products produced with an unanchored (①) or anchored RT primer (②) in the RT reaction at a low concentration of dNTPs (0.4 μM). In contrast, no significant differences in the signal intensities of the RT-PCR product are observed for 3' non-modified small RNAs (miRNAs) regardless of the dNTP concentration or primer (i.e. anchored or unanchored) used in the RT reaction. The ratio of PCR signal intensity is shown beneath each lane. Each assay was performed at least three times and the results of a representative assay are shown. N = any nucleotide base.

The detection of partially 2'-O-methylated sites in RNA species. The percentage of RNA substrates with a 2'-O-methylation site in total RNA detected is shown above each lane; the MeUA-RT/MeA-RT signal intensity ratio is indicated beneath each lane.