qPCR Methodology StandardizationFeatured
Development of standardized qPCR protocols for bacterial gene expression analysis, adopted as the standard by Heriot-Watt University's qPCR Core Facility.
Overview
During my PhD research at Heriot-Watt University (2012-2016), I developed and standardized advanced quantitative PCR (qPCR) methodologies for analyzing bacterial gene expression. This work focused specifically on examining starch metabolism operons in solventogenic Clostridium species across varied carbon sources.
The Challenge
Quantitative PCR is a powerful technique for measuring gene expression, but achieving reliable, reproducible results requires careful optimization and standardization. At the time, there was significant variability in qPCR protocols across different research groups, making it difficult to compare results and ensure data quality.
Key challenges included:
- Primer design and validation for genes in thermophilic and anaerobic bacteria
- Reference gene selection for accurate normalization
- Optimization of reaction conditions for difficult-to-amplify templates
- Standardization of data analysis methods to ensure consistency
The Approach
I developed a comprehensive qPCR workflow that addressed each of these challenges:
Primer Design and Validation
- Designed primers for multiple target genes involved in starch metabolism
- Validated primer efficiency and specificity using gradient PCR and melt curve analysis
- Established quality control criteria for primer acceptance
Reference Gene Validation
- Evaluated multiple housekeeping genes for expression stability across experimental conditions
- Used statistical algorithms (geNorm, NormFinder) to identify the most stable reference genes
- Established guidelines for reference gene selection in similar bacterial systems
Reaction Optimization
- Systematically optimized reaction conditions including primer concentrations, annealing temperatures, and template amounts
- Developed protocols for handling challenging templates from industrial fermentation samples
- Created standard operating procedures for RNA extraction and cDNA synthesis
Data Analysis Standardization
- Implemented the ΔΔCt method for relative quantification
- Established quality control thresholds for technical replicates
- Created templates for data analysis and visualization
The Impact
The standardized protocols I developed were subsequently adopted by Heriot-Watt University's qPCR Core Facility as their standard protocols for bacterial gene expression analysis. This ensured consistency across multiple research projects and provided a reliable foundation for future studies.
Key Outcomes
- Reproducibility: Improved inter-assay reproducibility from ~30% CV to less than 10% CV
- Efficiency: Reduced time required for assay optimization by providing validated protocols
- Adoption: Protocols adopted as standards by the university's qPCR Core Facility
- Publications: Contributed to peer-reviewed publications on bacterial metabolism
Skills Demonstrated
- Molecular biology techniques (qPCR, RNA extraction, primer design)
- Experimental design and optimization
- Data analysis and quality control
- Protocol development and documentation
- Scientific communication and knowledge transfer
Relevance Today
The skills I developed through this research continue to inform my work in commercial roles. Understanding the technical details of qPCR has been invaluable when working with customers at companies like Thermo Fisher Scientific and Roche Diagnostics, where I served as the go-to expert for complex qPCR sales.
The ability to bridge technical knowledge with practical application remains central to my approach in helping biotech companies develop and commercialize their products.