Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

Chang, Stewart T.; Thomas, Matthew J.; Sova, Pavel; Green, Richard R.; Palermo, Robert E.; Katze, Michael G.

Validation and temporal pattern of microRNA expression by qPCR. To validate other small RNA-Seq measurements, we performed qPCR on selected known microRNAs. Samples were derived from the same, increased-frequency experiment used to verify expression of the EPB41L2-encoded microRNA. The additional time points allowed a more precise estimation of the timing of expression patterns. Overall, we found that qPCR validated large fold changes in microRNA expression identified by small RNA-Seq, particularly 2-fold or greater upregulation (Fig. 5A to D; see also Fig. S5B to K in the supplemental material). For example, in the small RNA-Seq data set, we observed miR-4792 to be upregulated at 12 hpi and more highly upregulated at 24 hpi (Fig. 2B and 5A ). In the qPCR data set, this increase was found to occur more precisely between 12 and 16 hpi (Fig. 5A) . Likewise, miR-1248 was expressed at elevated levels at 24 hpi but not at 12 hpi in the small RNA-Seq data set (Fig. 5C ). In the qPCR data set, this increase was evident at an intermediate time point, 16 hpi (Fig. 5C) . The overall correspondence between qPCR and small RNA-Seq, particularly for upregulated microRNAs, was consistent with previous observations for mRNAs (18) .