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Related Concept Videos

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Related Experiment Video

Updated: Jan 16, 2026

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
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STALARD: Selective Target Amplification for Low-Abundance RNA Detection.

Daesong Jeong1,2, Chulmin Park1,2, Ilha Lee3,4

  • 1Laboratory of Plant Developmental Genetics, School of Biological Sciences, Seoul National University, Seoul, 08826, Korea.

Plant Methods
|September 30, 2025
PubMed
Summary

A new method called STALARD (Selective Target Amplification for Low-Abundance RNA Detection) enhances RNA isoform quantification. This rapid technique improves sensitivity for low-abundance transcripts, overcoming limitations of traditional methods.

Keywords:
Isoform quantificationLow-abundance RNAReverse transcription-quantitative real-time PCRSelective transcript enrichment

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Area of Science:

  • Molecular Biology
  • Genetics
  • Bioinformatics

Background:

  • Accurate RNA isoform quantification is crucial for understanding gene regulation.
  • Conventional RT-qPCR lacks sensitivity for low-abundance transcripts (Cq > 30).
  • Transcriptome-wide sequencing is costly and complex; isoform-specific qPCR faces primer efficiency bias.

Purpose of the Study:

  • To develop a sensitive and rapid method for quantifying low-abundance and alternatively spliced RNA isoforms.
  • To overcome the limitations of conventional RT-qPCR in detecting and quantifying specific transcript variants.

Main Methods:

  • Developed STALARD (Selective Target Amplification for Low-Abundance RNA Detection), a two-step RT-PCR method.
  • STALARD selectively amplifies polyadenylated transcripts with a known 5'-end sequence.
  • Utilized standard laboratory reagents for a rapid protocol (< 2 hours).

Main Results:

  • STALARD successfully quantified low-abundance transcripts, including VIN3, in Arabidopsis thaliana.
  • Detected known alternative splicing events for FLM, MAF2, EIN4, and ATX2 isoforms, even when conventional RT-qPCR failed.
  • Enabled consistent quantification of the low-abundance antisense transcript COOLAIR and revealed novel polyadenylation sites using nanopore sequencing.

Conclusions:

  • STALARD offers a sensitive, simple, and accessible method for isoform-level quantification of low-abundance transcripts with known 5'-end sequences.
  • The method is compatible with both qPCR and long-read sequencing, enhancing its versatility.
  • STALARD aids in analyzing transcript variants and discovering novel 3'-end structures.