Purification-free chemical conversion-coupled RT-PCR for ultralow-input detection and quantification of a6A-labelled RNA.

Nucleoside analogue-based RNA labelling provides powerful approaches for investigating RNA synthesis, turnover, and post-transcriptional regulation. However, conventional detection workflows often require chemical treatment, reagent removal, and repeated RNA purification, which can cause substantial sample loss and limit their application to ultralow-input samples. Here, we present a chemical conversion-coupled RT-PCR method that eliminates post-conversion RNA purification for the detection and quantification of N6-allyladenosine (a6A)-labelled RNA. Iodine-induced cyclization converts a6A into a cyclized adenosine derivative that generates characteristic cDNA mutation signatures during reverse transcription. By eliminating iodine removal, alkaline stabilization, and post-conversion RNA purification, the workflow enables reverse transcription directly from the chemical reaction mixture and reduces handling steps that may otherwise cause sample loss. Optimization of iodine treatment and reverse transcriptase compatibility identified Induro reverse transcriptase as suitable for quantitative analysis because it combined a high mutation rate with the highest estimated read-through efficiency across cyclized a6A sites. The method detected a6A-labelled RNA from femtogram-level inputs and supported mutation-rate-based quantification across a broad input range. Its applicability was further demonstrated by time-resolved tracking of IVT-generated mRNA carrying different poly(A) tails using ultralow amounts of total RNA. This workflow provides a sensitive platform for the detection and relative quantification of a6A-labelled RNA when sample availability is limited.