The RT at low dNTP concentrations followed by PCR (RTL-P) method is used for the detection of 2′-O-methylated sites in the 3′-end of RNA (21 (link)). For the detection of the modification of pi-snoRNAs, the total RNA was isolated using the Trizol reagent (Ambion) and 500 ng RNA was ligated to a 3′ RNA adapter using T4 RNA ligase (Takara). The ligation product was then reverse-transcribed using a low (0.4 μM) or high (40 μM) dNTP concentrations with or without anchored RT primers that were designed to anchor the modified nucleotide. The complementary DNA (cDNA) was subsequently amplified by PCR with specific primers under the same reaction conditions. The PCR products were then equally loaded and separated on 1.5% agarose gels, stained with GelRed dye (Biotium) and visualized by UV. It is notable that the RT-PCR product from the 3′ modification of piRNA was low when the RT reaction was done with an unanchored RT primer at a low concentration of dNTPs (0.4 μM). Otherwise, there was no differences in the product despite of the dNTP concentration or primers (anchored or unanchored) used in the RT reaction (20 (link)).