NEB TV Ep. 16 – Overview and Applications of qPCR

Introduce qPCR as a real-time DNA amplification monitoring technique that detects fluorescence signals during reaction progression. Describe two main detection approaches: DNA-binding dyes like SYBR Green and probe-based methods that generate signal through fluorophore separation during amplification.

Outline the experimental design comparing Luna master mixes with commercially available alternatives across multiple targets using five-fold dilution series, no-template controls, and triplicate runs. Document 18 different qPCR curves per target with two different users to generate a large dataset requiring new analysis methods.

Create a scalable data visualization by plotting PCR efficiency against delta Cq (difference between lowest template dilution and no-template control). Establish guideline parameters: 90-110% efficiency and delta Cq of three or greater to consolidate 18 wells of data per target into single data points.

Assign quality scores from 1-5 to each qPCR run based on curve sigmoidality and triplicate Cq tightness using methods adapted from published literature. Use the quality score to define dot size in the visualization, with larger dots representing higher quality scores.

Analyze performance of Luna Universal qPCR master mix versus other commercial DNA dye-based mixes across 18 unique genomic and cDNA targets run in triplicate by two users. Demonstrate that 86% of Luna experiments yielded high-quality results falling within acceptable parameters.

Confirm Luna qPCR kits work with diverse instruments regardless of passive reference dye requirements and cycling conditions. Explain that the blue inert reference dye improves well visualization on 96 and 384-well plates without inhibiting or interfering with qPCR detection.

Validate universal probe master mix for multiplex applications by testing up to three amplicons with different fluorophores detected on separate instrument channels. Optimize multiplex reactions by individually testing each amplicon separately first, then adjusting primer concentrations for targets with lower abundance while decreasing concentrations for high-abundance targets.