At this year’s American Society of Human Genetics annual meeting, Technology Networks caught up with Dr. Chelsea Pratt, biopharma segment manager at Bio-Rad Laboratories, who was exploring advances in digital polymerase chain reaction (PCR) technology and its potential to enhance gene expression analysis.
In this Q&A, Pratt discusses a recent study comparing multiplex droplet digital PCR (ddPCR) with traditional quantitative PCR (qPCR), highlighting key methodological differences, assay validation strategies and the advantages of ddPCR for detecting subtle changes in low-abundance targets. The discussion also explores practical considerations for labs adopting multiplex ddPCR and the areas of research where this technology could have the greatest impact.
Isabel Ely, PhD (IE):
Science Writer
Technology Networks
Isabel is a Science Writer and Editor at Technology Networks . She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Her doctoral research explored the role of dietary protein and exercise in optimizing muscle health as we age.
What key gaps in current standardized methods for ddPCR did this study aim to address?
Segment Marketing Manager
Bio-Rad
Dr. Chelsea Pratt is the biopharma segment marketing manager at Bio-Rad Laboratories. In this role, she works with leaders in the biotech and pharmaceutical industries to understand how Bio-Rad can accelerate drug discovery and development in new therapeutic areas.
To provide an example of data analysis and comparison to qPCR, highlighting the differences in data acquisition and analysis. The results were similar, with digital providing higher statistical significance.
Science Writer
Technology Networks
Isabel is a Science Writer and Editor at Technology Networks . She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Her doctoral research explored the role of dietary protein and exercise in optimizing muscle health as we age.
Can you walk us through the experimental design and why HeLa cells treated with cisplatin were chosen as the model system?
Segment Marketing Manager
Bio-Rad
Dr. Chelsea Pratt is the biopharma segment marketing manager at Bio-Rad Laboratories. In this role, she works with leaders in the biotech and pharmaceutical industries to understand how Bio-Rad can accelerate drug discovery and development in new therapeutic areas.
This model was chosen for its ease of implementation and thorough publication history. The genes chosen had previously been shown to have differential regulation in several publications, giving us an established protocol to use for technology comparisons.
Science Writer
Technology Networks
Isabel is a Science Writer and Editor at Technology Networks . She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Her doctoral research explored the role of dietary protein and exercise in optimizing muscle health as we age.
Multiplexing can sometimes lead to assay interference. What strategies did you use to minimize cross-reactivity and ensure robust results?
Segment Marketing Manager
Bio-Rad
Dr. Chelsea Pratt is the biopharma segment marketing manager at Bio-Rad Laboratories. In this role, she works with leaders in the biotech and pharmaceutical industries to understand how Bio-Rad can accelerate drug discovery and development in new therapeutic areas.
We used Bio-Rad PrimePCR assays, which have been tested for optimum performance at the same temperatures and protocols for each respective technology.
We validated the results by comparing single-plex results to multiplex results and had no difference in either technology.
Assay interference is a common concern with qPCR; however, we found that the robustness of the PrimePCR assays did not need any additional optimization in this experiment. Still, validation of multiplex results should always be confirmed before proceeding to complex experiments.
Science Writer
Technology Networks
Isabel is a Science Writer and Editor at Technology Networks . She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Her doctoral research explored the role of dietary protein and exercise in optimizing muscle health as we age.
What were the biggest differences you observed between qPCR and ddPCR in terms of sensitivity and precision for low-abundance targets?
Segment Marketing Manager
Bio-Rad
Dr. Chelsea Pratt is the biopharma segment marketing manager at Bio-Rad Laboratories. In this role, she works with leaders in the biotech and pharmaceutical industries to understand how Bio-Rad can accelerate drug discovery and development in new therapeutic areas.
ddPCR was able to measure low-abundance targets with higher precision and provide statistical significance of change in expression, where qPCR was not.
Science Writer
Technology Networks
Isabel is a Science Writer and Editor at Technology Networks . She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Her doctoral research explored the role of dietary protein and exercise in optimizing muscle health as we age.
In what types of studies or clinical applications do you see multiplex ddPCR having the greatest impact?
Segment Marketing Manager
Bio-Rad
Dr. Chelsea Pratt is the biopharma segment marketing manager at Bio-Rad Laboratories. In this role, she works with leaders in the biotech and pharmaceutical industries to understand how Bio-Rad can accelerate drug discovery and development in new therapeutic areas.
Multiplex ddPCR will be able to provide researchers with more reliable data for limited samples and genes that are only modestly elevated or reduced.
I see this having a large impact in neuroscience studies in research, as samples are extremely precious. It could also affect monitoring disease recurrence in patients with known and common mutations.
Science Writer
Technology Networks
Isabel is a Science Writer and Editor at Technology Networks . She holds a BSc in exercise and sport science from the University of Exeter, a MRes in medicine and health and a PhD in medicine from the University of Nottingham. Her doctoral research explored the role of dietary protein and exercise in optimizing muscle health as we age.
What advice would you give to labs deciding between qPCR and ddPCR for multiplex gene expression analysis?
Segment Marketing Manager
Bio-Rad
Dr. Chelsea Pratt is the biopharma segment marketing manager at Bio-Rad Laboratories. In this role, she works with leaders in the biotech and pharmaceutical industries to understand how Bio-Rad can accelerate drug discovery and development in new therapeutic areas.
Each technology can measure changes in gene expression, although ddPCR is capable of detecting less than two-fold changes, while qPCR cannot. Multiplexing with both technologies needs to be validated, comparing singleplex to multiplex results. Due to the partitioning done in ddPCR, amplification efficiency is less of a concern than with qPCR. However, optimized and validated PrimePCR assays showed that a robust design can reduce, and in this case, eliminate, additional assay optimization.
