by David Klowden
In January, 2020, a 35-year-old man who had recently visited Wuhan, China, was hospitalized for pneumonia in Washington.
It was the first confirmed case of COVID-19 in the United States.
The man survived, but many others were not so lucky. By March, over 100 people infected with the virus were dead. By May, that number had climbed to over 100,000.
His illness was a landmark in more ways than one – much of today’s research on the U.S. emergence of SARS-CoV-2 is built on the isolate known as WA1/2020, recovered from this single patient just north of Seattle. The WA1/2020 strain has also been used in the testing of vaccines, antivirals, and therapeutics for SARS-CoV-2.
Clinical variants in the pandemic’s first wave remained closely related to WA1/2020, with few nonsynonymous mutations. Researchers from the Department of Microbiology, Immunology and Biochemistry at the University of Tennessee Health Science Center in Memphis, TN, recognized this homogenous landscape of early strains as an ideal setup for teasing out the links between different disease phenotypes and individual viral mutations.
Using de-identified positive SARS-CoV-2 specimens collected from patients across four states in April 2020 and a mouse model capable of mirroring severe COVID-19 symptoms in humans, the researchers identified significant differences among four clinical isolates as compared to WA1/2020 in their replication, immune response, or pathology. One genetic isolate in particular conferred a lower median survival in infected mice.
To drill down to matching phenotypic and genotypic variations, they turned to multiplex assays for simultaneous gene and protein analysis using the Luminex® MAGPIX® System. For protein expression analysis, they opted for a 36-multiplex Invitrogen™ ProcartaPlex™ Mouse Cytokine/Chemokine Convenience Panel 1A immunoassay of mouse lung tissue from Thermo Fisher Scientific, and for gene expression, a 50-plex custom Invitrogen™ QuantiGene™ panel assay with a mix of biomarkers for viral and mouse RNA.
The technologies the Tennessee researchers used represent a sophisticated RUO large-scale, multiplexing technology for screening biomarkers (cytokines, chemokines, growth factors) implicated in cancer, infectious disease, and immunology. The Luminex platform leverages flow-cytometry–based instrumentation and Luminex xMAP® bead-based technology to produce results comparable to qPCR, with greater efficiency and throughput. The platform has been primarily used to study proteins using ProcartaPlex assays and not as frequently to study RNA, or, as in the Tennessee study, to analyze proteins and RNA simultaneously in the same experiment and from the same sample.
While the ability to perform multiplex studies of RNA along with proteins on Luminex systems isn’t widely known, it has clear advantages over qPCR and competitor technologies. It allows for highly targeted studies while greatly expanding the number of target parameters that can be analyzed at the same time, helping overcome the limitations caused by sample scarcity and variability across samples, and saving researchers on both the time and cost of analysis.
For years, Thermo Fisher has been creating custom panels for QuantiGene Plex multiplex RNA research like the panel used in the Tennessee study, but this year, for the first time, Thermo Fisher is introducing off-the-shelf panels for multiplex RNA analysis, making such investigations more accessible to more researchers.
Panels for the study of the immune response in mice, and cancer in both humans and mice, as well as toxicity in human samples launched in 2023. However, the new Invitrogen™ Immune Response 80-Plex Human QuantiGene™ Plex Panel for the study of human immunity is available now, offering researchers an off-the-shelf option for high-throughput, multiplex analysis of up to 80 genetic biomarkers to study all aspects of the immune response within one sample in just a single well of a 96- or 384-well plate.
Like the QuantiGene Plex custom panels, this new off-the-shelf immune response panel is tested for target combinability, interference, and cross-reactivity to provide a high level of validation and precision. The panel is used in combination with the QuantiGene Plex Assay Kit, which contains all the buffers, plates, and detection reagents necessary to perform the assay.
The panel is unique for its “true multiplexing” capability: this means researchers can measure 80 genes of interest and housekeeping genes together in the same well without cross-reactivity. The assay can also accommodate difficult sample types, such as degraded and cross-linked RNA in FFPE tissues and blood.
And while the panel is multiplex, it’s not complex. The assay employs an ELISA-like workflow with direct hybridization of transcripts to beads and transcript labeling (branched DNA signal amplification and PE readout), and it runs on a standardized platform: a 96-plate format compatible with the Luminex 200™, FLEXMAP3D®, and xMAP INTELLIFLEX® instrument systems. A 384-well format is also available on demand. Another key advantage of this innovative panel is rapid turnaround and fast delivery. The pre-validated, off-the-shelf panel ships from Thermo Fisher within 48 hours of an order. The off-the-shelf panel can further be customized with validated or alternative targets if needed and can be selected from Thermo Fisher’s inventory of over 22,000 genes.
The Immune Response 80-Plex Human QuantiGene™ Plex Panel, along with the other four off-the-shelf panels to be released later this year, represent the potential democratization of multiplexing for genetic biomarkers: empowering researchers with the ability to simultaneously study more samples and targets.
Learn more at thermofisher.com/quantigene»
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References
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Snohomish County man has the United States’ first known case of the New Coronavirus. The Seattle Times. March 11, 2020. Accessed July 24, 2023. https://www.seattletimes.com/seattle-news/health/case-of-wuhan-coronavirus-detected-in-washington-state-first-in-united-states/.
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United States coronavirus (covid-19) death toll surpasses 100,000. Centers for Disease Control and Prevention. May 28, 2020. Accessed July 24, 2023. https://www.cdc.gov/media/releases/2020/s0528-coronavirus-death-toll.html.
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