Qubit Fluorometric Quantification System Sample Data

Accuracy and precision of Qubit Fluorometers. Accuracy was assessed as average deviation from true concentration using the Qubit dsDNA HS Assay, while precision was evaluated as coefficient of variation (CV) across replicates of various concentrations using the Qubit dsDNA BR Assay. The low deviation percentages demonstrate the accuracy of the Qubit Flex and Qubit 4 Fluorometers, while the low CV percentages demonstrate their precision, especially the Qubit Flex model. Of the three instruments tested, the competitor’s instrument had the lowest accuracy and precision.

Qubit dsDNA accuracy and sensitivity vs UV absorbance. Ten replicates of lambda DNA at known concentrations from 0.01 to 10 ng/μL were assayed using the Qubit dsDNA HS Assay on the Qubit Fluorometer (blue bars) according to the standard kit protocol. The same concentrations of DNA were measured via UV absorbance in 10 replicates using a microvolume spectrophotometer (blue bars), and results were compared for both accuracy and precision. Each bar represents the average of 10 replicates, while error bars (almost indiscernible for Qubit measurements) represent their standard deviations. The x-axis scale represents the known concentrations of DNA in the starting samples, before dilution in the Qubit assay tubes, while the y-axis scale shows the measured concentrations. The Qubit Fluorometer measurements were more accurate (y » x), sensitive (low concentrations, inset), and precise (much smaller error bars) than the UV absorbance measurements.

Qubit microRNA assay accuracy, precision, and sensitivity. Six experiments were run to test the accuracy and precision of the Qubit microRNA assay with pure siRNA; results of a typical experiment are shown here. In this experiment, eight replicates of GAPDH siRNA in known concentrations from 0.1 μg/mL to 12 μg/mL were measured using the Qubit microRNA Assay Kit with the Qubit Fluorometer and using A₂₆₀ measurements on the NanoDrop ND-1000 Spectrophotometer. The detection limit for the NanoDrop instrument is reported to be 1.5 μg/mL; lower siRNA concentration results are shown for comparison. Accuracy for the Qubit microRNA Assay was within 15% of expected. CVs (1 standard deviation/average of 8 data points) were 0.63% to 2.9%. For all six experiments, the Qubit microRNA assay accuracy was within 15% of expected, and CVs were <5%.

Qubit microRNA assay accuracy and breadth. Concentrations of pure siRNA and miRNA samples were determined by optical density on a PerkinElmer® spectrophotometer at concentrations yielding an A₂₆₀ of 0.3–0.6. Samples were then diluted and tested with the Qubit microRNA Assay at four different known concentrations. Results show accurate quantification of (A) four different single-stranded siRNA molecules and (B) two double-stranded siRNAs and two double-stranded miRNAs.

Selectivity of the RNA IQ assay reagents for large and small RNA. Triplicate samples containing 100 ng/µL rRNA (E. coli) and varying amounts of siRNA (0–50 ng/µL) were assayed with the Qubit RNA IQ assay on the Qubit 4 Fluorometer. Graphs show (A) relative fluorescence units (RFUs) and (B) IQ scores for these samples. The data shows that the dyes are specific to large (like rRNA) and small (like siRNA) RNA respectively, and that IQ scores are strongly correlated with the percentage of large RNA in the sample.

rRNA degradation by RNase A over time, measured by the Qubit RNA IQ Assay. Triplicate samples of 100 ng/µL rRNA solutions were incubated with different doses of RNase A in the final assay solution containing multiplexed dyes and assay buffer. (A) Higher doses of RNase A caused faster degradation of RNA over 60 minutes as reflected by its RNA IQ score. (B) Exposure to 10 fM RNase A caused a gradual decline in large RNA fluorescence over the hour and a corresponding increase in small RNA fluorescence. (C) With exposure to 100 fM RNase A, the changes occurred more quickly.

RNA IQ is consistent with RT-qPCR results while Bioanalyzer™ RNA integrity number (RIN) is not. Total RNA (isolated from human liver) was heat-treated at 75°C for varying durations and analyzed using the Agilent™ Bioanalyzer™ system and Qubit RNA IQ Assay. RT-qPCR analysis was also performed using RETROScript reverse transcriptase and TaqMan hHIF1α and hGAPDH assays. (A) Data from the Bioanalyzer system show rapidly decreasing rRNA peaks over time, suggesting deteriorating RNA integrity. (B) Comparison of RIN (black dots) and RNA IQ (gray triangles), including more detailed results at the 40 min time point, shows disagreement, with RIN decreasing rapidly but RNA IQ largely stable over time. (C) RT-qPCR results were also stable over time, in agreement with RNA IQ but not RIN.

Accurate determination of protein load from complex protein mixtures. The Qubit Protein BR Assay and a standard Bradford assay were used to determine the protein concentration of lysates from several mammalian cell types: 293T, A549, HepG2, HeLa, and iPSCs. Lysates were separated on an Invitrogen NuPAGE 4–12% Bis-Tris Mini Protein Gel and labeled with No-Stain Protein Labeling Reagent. (A) Gel image was acquired on the iBright FL1500 Imaging System and (B) normalization factors were determined using Invitrogen iBright Analysis Software. The coefficient of variation (CV) of the Qubit Protein BR Assay was appreciably lower than that of the Bradford assay.