PABLO-QA: A sensitive assay for quantifying monophosphorylated RNA 5′ ends

Generated by RNA deprotection or cleavage, 5′ monophosphates trigger RNA degradation in all organisms. Here we describe PABLO-QA (Phosphorylation Assay By Ligation of Oligonucleotides and Quantitative Amplification), a sensitive, low-cost procedure for determining the percentage of specific RNA 5′ ends that are monophosphorylated from their ability to undergo ligation to an oligonucleotide. Comparison to a cognate internal standard and a fully monophosphorylated control allows precise quantification of monophosphorylated 5′ termini by RT-PCR, enabling the analysis of transcripts undetectable by blotting. For complete details on the use and execution of this protocol, please refer to Richards and Belasco (2021).