A reliable and quick method for screening alternative splicing variants for low-abundance genes

Area of Science:

• Molecular Biology
• Genetics

Background:

• Alternative splicing (AS) is a crucial eukaryotic process, yet identifying AS events, particularly in low-abundance genes, remains challenging.
• Existing methods like expressed sequence tags (EST), microarrays, and RNA-seq have limitations, including poor detection of rare transcripts and the need for validation.

Purpose of the Study:

• To establish a reliable, cost-effective, and labor-efficient method for identifying alternative splicing events, with a focus on low-abundance genes.
• To overcome the limitations of current AS detection technologies.

Main Methods:

• Developed a two-step reverse transcription (RT) followed by nested PCR strategy using gene-specific primers (GSPs).
• Step 1: GSPs amplify specific gene segments.
• Step 2: Multiple rounds of nested PCR screen for and confirm AS variants, including novel ones.

Main Results:

• Successfully identified three novel splicing variants for the bdnf, trkc, and glb-18 genes (GenBank Accession Nos. HM623886, JF417977, HM623888).
• The RT-nested PCR method demonstrated reliability, simplicity, and cost-effectiveness.
• The technique efficiently detected rare transcripts and unknown splicing variants.

Conclusions:

• An efficient RT-nested PCR method using gene-specific primers was developed for identifying both known and novel alternative splicing variants.
• This approach effectively addresses the limitations of existing methods for detecting rare transcripts.
• The technique holds promise for uncovering AS variants in diseases such as cancer and neurodegeneration, advancing research into splicing-related disorders.