lfe sciences
naica® Droplet Chip Digital PCR System Accurately Quantifies R613X Transcription Levels in Dravet Syndrome Scn1a Nonsense Mutation Model
2024-04-28
Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. SCN1A nonsense mutations are found in approximately 20% of patients, and SCN1A R613X mutations have been identified in multiple patients. Researchers from the Sagol School of Neuroscience, the Department of Human Molecular Genetics and Biochemistry, and the Goldschleger Eye Institute at Tel Aviv University, Israel, published an article titled "Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome" on the bioRxiv platform.
The authors constructed a Dravet mouse model containing a nonsense Scn1a mutation, in which the heterozygous Scn1a R613X mutation exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature death, recapitulating the core epilepsy phenotype of Dravet. In this study, the authors used the naica® Droplet Chip Digital PCR System for allele-specific quantification of WT Scn1a and R613X mRNA.
Application Highlights:
▶ The naica® Droplet Chip Digital PCR System accurately quantified the content of WT Scn1a and R613X mRNA alleles in the Dravet mouse model.
▶ In heterozygous animals, the naica® Droplet Chip Digital PCR System quantified stable R613X transcript levels at 8.9±0.9% of wild-type, indicating that transcripts of the mutant allele undergo nonsense-mediated RNA decay (NMD) in cortical tissue.
To quantitatively assess the levels of R613X transcripts in the cortex, the naica® Droplet Array Digital PCR System was used to specifically detect WT Scn1a and the R613X allele.
Results:
The naica® Microdroplet Chip Digital PCR System did not detect the R613X allele in cortical tissues of WT mice, confirming the specificity of the method (Figure A). The WT allele was detected at approximately half the level in Scn1aWT/R613X tissues compared to WT cortex, as expected for heterozygous animals (Figure B). In heterozygous animals, stable mutant R613X transcripts were 8.9±0.9% (Figure C), much lower than the expected 50% level, indicating that transcripts of the mutant allele undergo strong nonsense-mediated decay (NMD) in cortical tissues.
▲Figure. Reduced Scn1a mRNA expression in the cortex of Scn1aWT/R613X mice. A. Cortical tissue from WT mice showed no detectable R613X allele transcripts, demonstrating the specificity of detection of this allele. B. In cortical tissue from Scn1aWT/R613X mice, the WT Scn1a allele was 48.8% of that in the WT cortex. C. The ratio of R613X to WT alleles showed that the average steady-state level of the Scn1a R613X allele in Scn1aWT/R613X animals was 8.9±0.9% of the WT allele.
Original link is as follows: https://doi.org/10.1101/2023.02.01.526678
The naica® six-channel digital PCR system of Stilla Technologies, France, is based on the Crystal droplet chip digital PCR technology, which automates droplet generation and amplification. Each sample well can detect 6 fluorescent channels, intelligently identify droplets and perform quality control, and obtain the absolute copy number concentration of at least 6 target genes within 3 hours.
