STALARD: Selective Target Amplification for Low-Abundance RNA Detection

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.