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.

High-sensitivity microvolume UV absorption spectrometry for routine analysis of small-volume biological samples.

Substantial improvement of microvolume UV absorption spectrometry in sensitivity, robustness and ease of operation was achieved for routine biological applications. A unique microtubing-based absorption cell (208 µm internal diameter) featuring enhanced light transmission with a liquid core waveguide technique provided dramatically enhanced absorption sensitivity, proportional to the extended path length (50 mm, from the typical 1 mm), while robust measurement performance was attained by implementation of preventive measures against bubble trapping along the light path. For pBR322 plasmid DNA, absorbance at 260 nm was reliably measurable down to 0.1 ng/µl with repeatability typically 2-3% RSD. The detection limit was 0.03 ng/µl dsDNA, far lower than the current state-of-the-art âˆ¼1 ng/µl. Sample consumption for each measurement was 2.4 µl. Automated operation implemented for the first time in microvolume spectrophotometry facilitated the ease in handling with small-volume biological samples.

Accurate quantification of supercoiled DNA by digital PCR.

Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.