STALARD: Selective Target Amplification for Low-Abundance RNA Detection

Background Accurate quantification of transcript isoforms is critical for understanding gene regulation. However, conventional reverse transcription-quantitative real-time PCR (RT-qPCR) has limited sensitivity for low-abundance transcript isoforms, as quantification cycle (Cq) values above 30 are often considered unreliable. While transcriptome-wide analyses can address this limitation, they require costly deep sequencing and complex bioinformatics. Results To overcome the sensitivity limitations of conventional RT-qPCR for detecting low-abundance and alternatively spliced transcripts, we developed STALARD (Selective Target Amplification for Low-Abundance RNA Detection), a rapid (<2 hr) and targeted two-step RT-PCR method using standard laboratory reagents. STALARD selectively amplifies transcripts sharing a common 5′-end, enabling efficient quantification of low-abundance isoforms. When applied to Arabidopsis thaliana, STALARD successfully amplified the low-abundance VIN3 transcript to reliably quantifiable levels. Amplification of FLM, MAF2, EIN4, and ATX2 isoforms by STALARD reflected known splicing changes during vernalization, including cases where conventional RT-qPCR failed to detect relevant isoforms. STALARD also enabled consistent quantification of the extremely low-abundance antisense transcript COOLAIR, resolving inconsistencies reported in previous studies. In combination with nanopore sequencing, STALARD further revealed novel COOLAIR polyadenylation sites not captured by existing annotations. Conclusion STALARD provides a sensitive, simple, and accessible method for isoform-level quantification of low-abundance transcripts. Its compatibility with both qPCR and long-read sequencing makes it a versatile tool for analyzing transcript variants and identifying previously uncharacterized 3′-end structures.