Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Accurate quantification of alternative mRNA splicing is crucial for understanding gene expression.
• Existing methods for quantifying splice variants lack sensitivity, precision, and reproducibility.
• Human telomerase reverse transcriptase (hTERT) has several alternatively spliced forms implicated in cellular processes.

Purpose of the Study:

• To develop and describe a novel droplet digital PCR (ddPCR) method for precise quantification of alternatively spliced hTERT mRNA variants.
• To enable direct quantitative comparison of multiple hTERT splice forms from a single ddPCR reaction.

Main Methods:

• Utilized droplet digital PCR (ddPCR) with a single primer pair and two probes specific for hTERT.
• Developed a novel analysis method to quantify four distinct alternatively spliced hTERT mRNA variants (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+).
• Eliminated the need for internal standards or multiple primer sets, avoiding variations in PCR amplification efficiency.

Main Results:

• Successfully quantified four alternatively spliced forms of hTERT mRNA with high accuracy and precision using the developed ddPCR method.
• Demonstrated the ability to directly compare different hTERT splice variants within a single ddPCR experiment.
• Showcased the method's independence from standard curves and its robustness against differential amplification efficiencies.

Conclusions:

• The described ddPCR method provides a sensitive, precise, and reproducible means to quantify alternatively spliced hTERT mRNA.
• This straightforward approach simplifies the analysis of alternative splicing for hTERT and potentially other genes.
• The method offers a significant advancement for gene expression studies involving splice variant quantification.