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Updated: Aug 28, 2025
High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes
Published on: March 24, 2015
A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs
Daniel J Browne1, Ashton M Kelly1, Jamie L Brady1
1Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.
Insights
We developed a cost-effective, high-throughput screening (HTS) assay using reverse transcription-quantitative PCR (RT-qPCR) to measure immune responses from limited peripheral blood mononuclear cells (PBMCs). This sensitive method significantly reduces costs and reagent use for immune monitoring.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Current immunoassays for immune effector function, like flow cytometry and ELIspot, require high cell numbers and expensive reagents, limiting comprehensive analyses and high-throughput screening (HTS).
- There is a need for sensitive, cost-effective assays capable of quantifying immune markers from limited peripheral blood mononuclear cells (PBMCs).
Purpose of the Study:
- To develop and validate a sensitive, specific, and cost-optimized HTS assay for quantifying immune surrogate markers from low cell numbers of PBMCs.
- To reduce the cost and cell consumption of current immune monitoring assays while maintaining high sensitivity and accuracy.
Main Methods:
- Development of a reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay.
- Systematic optimization of reagent volumes and concentrations to miniaturize the assay.
- Validation of the assay's HTS suitability, analytical sensitivity, and diagnostic accuracy.
Main Results:
- The developed RT-qPCR assay significantly reduces costs by almost 90% compared to standard methods.
- The assay meets HTS uniformity and signal variance standards and demonstrates single-cell analytical sensitivity.
- It accurately delineates immune responses from as few as 50,000 PBMCs and achieves diagnostic sensitivity comparable to ELIspot.
Conclusions:
- The cost-optimized RT-qPCR assay provides a sensitive and specific HTS tool for quantifying immune responses from limited PBMCs.
- This assay has widespread applicability in preclinical and clinical studies where sample availability and cost are critical considerations.
- The technique offers a valuable alternative for immune monitoring, enhancing the scope of research and diagnostics.
Abstract:
Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.
