After performing microarray profiling of RNA from cells exposed or not to a chimeric murine virus known as XMRV, Mohan, et al. suggest that microarray analysis "clearly demonstrates that microRNAs are regulated during XMRV infection," specifically miR-193a-3p and a putative miRNA called E1245. However, the lack of biological or technical replicates, statistical analysis, and validation assays might make it difficult for some to accept this conclusion.
Presenting PCA in Figure 2, the authors state that "the overall miRNA expression profile in LNCaP and DU145 cell lines form a tight cluster suggesting minimal variation due to time and infection status of these two cell types, while the PBLs and MDM profiles are more spread, indicating the global miR levels being affected both due to time and infection status." The final assertion seems to be somewhat inconsistent with Figure 2, showing that infected and uninfected PBL and MDM generally pair up based on time point, not infection status. A slightly different conclusion might be that cancer cell lines, being relatively clonal, tend to display less miRNA variation during culture than heterogeneous populations of primary blood cells, and that the presence of the chimeric murine virus has no readily apparent effect. This is also clear from the heatmap in Figure 3. Technical processing differences between the time points could be an equally valid explanation for apparent time point-associated expression differences.
Several clarifications would be much appreciated and would help me (and perhaps other readers) to understand the study better:
1. Because no statistical evidence of differential expression was found, the authors determined the difference between mock and infected conditions for each group of cells at the 6, 24, and 48 hour time points and report a single, undefined "abs" value in the Figure 5 table. What is the "abs" value? Is it the average difference between log(2) values for infected and uninfected samples for all time points? If so, it would be useful to know the direction of regulation as well as how much this difference varied between the time points. Differences (at 6, 24, and 48 hours) of 2, 2.2, and 2.1 average 2.1, but so do 0.1, 6.1, and 0.1. Some might consider the former case to be more convincing than the latter. A small amount of additional information could greatly strengthen the authors' conclusions.
2. In Figure 3, it is not clear what the authors mean by including "all microRNAs with standard deviation over 1". Further clarification would be useful. Also, although miR-193a-3p is depicted, where is the proprietary putative miRNA "E1245" in the heatmap?
3. The utility of microarray studies to the scientific community is greatly enhanced by availability of the data; hence, most journals, including PLoS ONE, mandate data submission to public databases such as GEO or ArrayExpress. The authors are strongly encouraged to upload their raw and processed array data as per journal policy (see http://www.plosone.org/st... and http://www.plosone.org/st... .)