A novel qPCR assay for viral encoded microRNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that postranscriptionally regulate viral and host gene expression. Reliable and simple assays for detecting and analyzing miRNAs during viral infections are critical for clinical and research purposes. A highly sensitive quantitative real-time PCR (qPCR) assay was developed. This approach, using a generic hydrolysis probe, detects and quantifies miRNAs in cell culture and in clinical samples obtained from patients. The assay is based on preparation of cDNA libraries by polyadenylation of total RNA and reverse transcription, followed by detection of specific miRNAs in the cDNA library by qPCR. The qPCR test was highly sensitive and specific, distinguishing between miRNAs that differ by as little as a single nucleotide with remarkable reproducibility. When applied to clinical samples the assay could detect the differential expression of EBV encoded miRNAs in peripheral blood cells of healthy EBV carriers and patients with acute EBV infection, which makes it a powerful tool for the study of differential expression of miRNAs in health and during viral infections.