Precise RNA Quantification by Counting Individual RNA Molecules Using High-Sensitivity Capillary Flow Cytometry.

Precise determination of ribonucleic acid (RNA) concentration without the need for calibration was pursued by sequence-specific counting of individual RNA molecules. This approach eliminates the reverse transcription (RT) step required for polymerase chain reaction (PCR)-based quantification, which may hamper accurate measurements owing to uncertain yields of RT reactions. Target RNAs were tagged with a number of fluorescent oligonucleotide probes with complementary sequences. Tagged RNAs were exhaustively counted one by one using a high-sensitivity capillary-based flow cytometric setup. MS2 viral RNA was quantified as a model RNA for which essential parameters, including probe numbers, probe concentration, and hybridization conditions, were optimized for the best performance. Using 70 oligonucleotide probes, MS2 RNA was quantified with 2.0% relative standard deviation, and its validity was assessed by comparison with other RNA quantification methods such as droplet digital PCR and UV spectrophotometry. The observed comparability indicated that the proposed method is unlikely to have a substantial bias. It works for a substantially lower-level RNA than UV and avoids the potential variability in the yield of the RT reaction of RT-qPCR. Therefore, the proposed method could be a valuable addition to current methods and could be further developed as a standard reference method for RNA quantification.