A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

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.

The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

“…All samples were primed with 37.5 ng of random hexamers and 10 mM dNTPs at 65 • C for 5 min and then 4 • C for 1 min. Reverse transcription was then performed using the SuperScript IV ™ reverse-transcriptase (SSIV) for 10 min at 23 • C, 10 min at 50 • C, and 10 min at 85 • C. SSIV concentration was assessed at 20 U (20 units/µL RNA) reactions compared with 5 U reactions (Figure S2) as previously described [41]. All subsequent RNA samples were reverse transcribed at 30 ng/µL (Figure S3) using 5 U reactions in 15 µL total volume reactions for test conditions.…”

Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria

“…The critical effector molecule of adaptive immunity to sporozoite challenge appears to be Interferon-gamma (IFN-γ) released by CD8 + T cells 34 , 55 , and Th1 CD4 + T cells secreting IFN-γ and Interleukin-2 (IL-2) 32 , 33 , 56 . The mRNA expression profiles of IFN-γ and many other rapidly produced and secreted cytokines are relatively highly correlated to protein production 43 . Therefore, transcriptomic quantification of host-cytokine responses will inform functional efforts to understand the immunological response following vaccination.…”

“…Extracted mRNA was quantified using a NanoPhotometer® N60 (Implen, München, Germany). RNA (0.4 μg) was then converted to cDNA using the SuperScript™ IV First-Strand Synthesis System (Invitrogen) in 10 μL total volume reactions with random hexamers only with the following modifications from the manufacturer's protocol: cDNA synthesis was conducted with the SuperScript™ reverse transcriptase at half the manufacturers recommended concentration (10U/µL RNA ), as previously described 43 .…”

“…qPCR was performed using ssoAdvanced SYBR® SuperMix (BioRad) following the manufacturer's recommendations (hot start 2 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C) 18 . Reactions were run at 5 μL total volume amplifying 1 μL sample, as previously described 43 . Reactions were measured by QuantStudio 5 Real-Time PCR Machine running QuantStudio Design and Analysis Software (v1.4.3, Applied Biosystems), using technical triplicates and no template negative controls.…”

Technical pitfalls when collecting, cryopreserving, thawing, and stimulating human T-cells

“…The length of time cells are incubated in the presence of the stimulant can influence the number and intensity of responsive cells. Six-hour long incubations are frequently reported ( 94 , 95 ), however, longer incubations are also common ( 96 ). Overnight incubations have been reported to increase antigen immunogenicity ( 97 ).…”