| title | Replication Study | ||
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| label | replication_page |
Reproducibility is an essential principle of the scientific method for acquiring knowledge. Repeatability in the same study by the same researchers is distinguished from replicability by independent researchers using the same methodology. Barkley noticed a lack of methods for reproducing representative images and proposed virtual nanoscopy as a method to assess the replicability of observations evidenced by representative images. Results should be observed again with a high degree of reliability, therefore the original claim should be evident within a sample of hundreds of cells. To test this, Barkley challenged the claim that reovirus remodels the ER [@doi:10.1128/mBio.01253-18]. Most evidence, including key findings, were representative images without quantitation (), which is problematic given the inability to communicate population variation and uncertainty. The study concluded that reovirus induced fragmented, collapsed, and aggregated ER elements, shown in images of infected cells expressing mCherry-KDEL. This fluorescent ER reporter was expressed using plasmid transfection or with engineered cell lines. Barkley designed the replication study using the transfected-infected cell model, and stable cells were used for cross-validation. In the transfected-infected model, plasmid transfection was followed by reovirus infection, then the cells were fixed and immunostained for ER (calreticulin) and viral (µNS) antigens. Therefore, the calreticulin channel shows the ER of all cells, reovirus µNS shows the viral factories of infected cells, and mCherry-KDEL shows the ER of transfected cells. From a sample of hundreds of cells, putative virus-induced ER remodelling should be detectable in the calreticulin and mCherry-KDEL channels between infected and uninfected cells. Heterogeneity was expected in the population due to viral permissivity and incomplete plasmid transfection efficiency, and coincidence of µNS and mCherry was considered desirable in this context. Another confounding variable was interferon induction from liposomal transfection reagents [@doi:10.1089/jir.1998.18.947] and plasmid DNA.
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