无需纯化步骤的化学转化-实时逆转录聚合酶链反应（RT-PCR）技术，可实现对a6A标记RNA的超低输入量检测与定量分析。

Abstract

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 N 6 -allyladenosine (a 6 A)-labelled RNA. Iodine-induced cyclization converts a 6 A 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 a 6 A sites. The method detected a 6 A-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 a 6 A-labelled RNA when sample availability is limited.