Automation and miniaturization of high-throughput qPCR for gene expression profiling

Area of Science:

• Molecular Biology
• Genomics
• Laboratory Automation

Background:

• Quantitative PCR (qPCR) is a standard laboratory technique for gene expression analysis.
• Previous work demonstrated partial automation of gene expression workflows using Mosquito HV and BioMark HD.
• Full automation and miniaturization potential of this workflow remained unexplored.

Purpose of the Study:

• To fully automate a gene expression workflow using Mosquito HV genomics software.
• To evaluate the impact of reaction miniaturization on data quality and reproducibility.
• To assess the performance of automated gene expression panels for immunology and pregnancy.

Main Methods:

• Scripting of each gene expression workflow step on Mosquito HV genomics software.
• Automated runs using Mosquito HV with BioMark HD, testing full reaction (FR) and 1.5x, 2.5x, 5x miniaturization.
• Comparison of automated FR replicates with manual processing for reference RNA.
• Analysis of biological RNA samples from 45 individuals on immunology and pregnancy panels using FR and 1.5x conditions.

Main Results:

• Successful amplification was achieved with FR and 1.5x miniaturization; 2.5x and 5x showed suboptimal or no amplification.
• Strong positive correlation observed between manual and automated workflows for reference RNA.
• Automated immunology and pregnancy panels (FR and 1.5x) achieved >70% success rate on 45 individual samples.
• Significant positive correlation between FR and 1.5x conditions for both gene expression panels.

Conclusions:

• Full automation of gene expression workflows using Mosquito HV is feasible.
• 1.5x miniaturization with Mosquito HV maintains data quality and reproducibility.
• Automated gene expression analysis is robust and suitable for large-scale sample processing.