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Background <p>Accurate quantification of RNA 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. Moreover, isoform-specific qPCR is often confounded by differential primer efficiency when comparing similar transcripts.</p> Results <p>To overcome the sensitivity and amplification bias limitations of conventional RT-qPCR for detecting known low-abundance and alternatively spliced transcripts, we developed STALARD (<i>Selective Target Amplification for Low-Abundance RNA Detection</i>), a rapid (&lt; 2&#xa0;h) and targeted two-step RT-PCR method using standard laboratory reagents. STALARD selectively amplifies polyadenylated transcripts sharing a known 5′-end sequence, enabling efficient quantification of low-abundance isoforms. When applied to <i>Arabidopsis thaliana</i>, STALARD successfully amplified the low-abundance <i>VIN3</i> transcript to reliably quantifiable levels. Amplification of <i>FLM</i>, <i>MAF2</i>, <i>EIN4</i>, and <i>ATX2</i> 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 <i>COOLAIR</i>, resolving inconsistencies reported in previous studies. In combination with nanopore sequencing, STALARD further revealed novel <i>COOLAIR</i> polyadenylation sites not captured by existing annotations.</p> Conclusion <p>STALARD provides a sensitive, simple, and accessible method for isoform-level quantification of low-abundance transcripts that share a known 5’-end sequence. 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, provided that isoform-specific 5′-end sequences are known in advance.</p>

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STALARD: Selective Target Amplification for Low-Abundance RNA Detection

  • 期刊论文

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  • 发表日期：

    2025年9月29日

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  • Daesong Jeong,
  • Chulmin Park,
  • Ilha Lee

摘要

Background

Accurate quantification of RNA 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. Moreover, isoform-specific qPCR is often confounded by differential primer efficiency when comparing similar transcripts.

Results

To overcome the sensitivity and amplification bias limitations of conventional RT-qPCR for detecting known low-abundance and alternatively spliced transcripts, we developed STALARD (Selective Target Amplification for Low-Abundance RNA Detection), a rapid (< 2 h) and targeted two-step RT-PCR method using standard laboratory reagents. STALARD selectively amplifies polyadenylated transcripts sharing a known 5′-end sequence, 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 that share a known 5’-end sequence. 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, provided that isoform-specific 5′-end sequences are known in advance.