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Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA

Department of Physiology and Pharmacology, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4Z6, Canada
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Methods Protoc. 2021, 4(1), 13; https://doi.org/10.3390/mps4010013
Received: 14 January 2021 / Revised: 5 February 2021 / Accepted: 9 February 2021 / Published: 11 February 2021
Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5′ non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function. View Full-Text
Keywords: rhinovirus; airway epithelium; viral replication; negative strand rhinovirus; airway epithelium; viral replication; negative strand
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MDPI and ACS Style

Wiehler, S.; Proud, D. Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA. Methods Protoc. 2021, 4, 13. https://doi.org/10.3390/mps4010013

AMA Style

Wiehler S, Proud D. Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA. Methods and Protocols. 2021; 4(1):13. https://doi.org/10.3390/mps4010013

Chicago/Turabian Style

Wiehler, Shahina, and David Proud. 2021. "Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA" Methods and Protocols 4, no. 1: 13. https://doi.org/10.3390/mps4010013

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