Materials and Methods
DeNovix RNA Assay
A series of dilutions of E. coli total RNA was prepared in DEPC water. The assay working solution was prepared by mixing 4 mL of the assay buffer with 20 µL of the dye. For each sample, 190 µL of the working solution was added to a thin-walled, clear UV-transparent 0.5 mL PCR tube (DeNovix cat #TUBE-PCR-0.5-500). 10 μL of RNA was added to each tube for samples with concentrations between 0.5 ng/μL and 100 ng/μL. For the samples with a concentration less than 0.5 ng/uL, volumes were adjusted to use 180 μL working solution with 20 μL of RNA. Measurements of concentrations above 100 ng/μL were made using volume adjustments so that the total mass did not exceed 1500 ng (e.g. 3 µL of 500 ng/µL in 197 µL of working solution).
Reaction solutions were mixed and incubated at room temperature for 10 minutes while protected from ambient light. Five replicate measurements of each sample were taken to assess linearity.
The linear response of measured RNA as a function of expected concentration in the full linear range is presented in Figure 1. The range of the assay can successfully be extended past both the upper and lower limits, and extension has impact on linearity.
Thermo Fisher Qubit™ Broad Range (BR) and High Sensitivity (HS) RNA Assays
A Thermo Fisher Qubit™ 4 fluorometer was used to test the same samples as the DeNovix RNA Assay with the Thermo Fisher Qubit™ BR and HS kits to minimize dilution differences. The Thermo Fisher Qubit™ BR kit (Q10210) (Lot #2086147) was tested across the published range of 20 ng – 1000 ng. Likewise, the Thermo Fisher Qubit™ HS kit (Q32852) (Lot #2113240) was tested over the published range of 5 ng – 100 ng. All manufacturers instructions for sample handling were followed for each kit.
Comparison with Thermo Fisher Qubit™ RNA Assays
The DeNovix RNA Assay covers a greater dynamic range than the combined range of the Thermo Fisher Qubit™ BR RNA and Qubit™ HS kits. Figure 1 compares the DeNovix RNA Assay dynamic range to the Thermo Fisher Qubit™ BR and Qubit™ HS RNA assays.
Figures 2 and 3 below demonstrate that the DeNovix RNA Assay enables measurement of RNA through a range of 0.5 ng (20 µL of 0.25 ng/μL) – 1500 ng (1 µL of 1500 ng/µL) by varying mass and volume of samples. The assay performance is linear over the range analyzed with an exceptional R2 value.
Table 1 lists the performance data that has been graphed in Figures 2 and 3. Five replicates were taken for each sample. The values in the expected category are based upon spectrophotometric analysis of each dilution to minimize the influence of pipetting errors.
|RNA Assay Performance Data|
measured on DS-11 FX+
|Thermo Fisher Qubit™ BR Assay measured on Qubit™ Fluorometer||Thermo Fisher Qubit™ HS Assay measured on Qubit™ Fluorometer|
|1500||1449.879||0.772||Out of Range||Out of Range||Out of Range||Out of Range|
|1000||990.358||0.426||1052||3.454||Out of Range||Out of Range|
|500||520.106||0.272||610.8||2.888||Out of Range||Out of Range|
|250||245.554||2.556||299.6||1.284||Out of Range||Out of Range|
|0.25||0.160||3.483||Out of Range||Out of Range||0.334||2.445|
|0||0||0||Out of Range||Out of Range||Out of Range||Out of Range|
The DeNovix RNA Assay enables specific, highly sensitive RNA quantification across a wide dynamic range. Compared to the Thermo Fisher Qubit™ assays, the DeNovix RNA assay requires only a single kit to cover a wider concentration range with highly linear, reproducible data. Researchers benefit from a greater proportion of samples fitting within the assay limits, decreasing the requirement to dilute or concentrate samples and eliminating the time and expense of repeating assays.
DeNovix Fluorescence Assays
DeNovix offers a range of fluorescence quantification assays for DS-11 Series fluorometers and other compatible instruments. All assays offer sensitive and specific quantification for either dsDNA or RNA samples in the presence of contaminants.
|DeNovix Fluorescence Assay||Range|
|dsDNA Broad Range||0.1 – 2000 ng/µL|
|dsDNA High Sensitvity||5 pg/µL – 250 ng/µL|
|dsDNA Ultra High Sensitivity||0.5 – 300 pg/µL|
|RNA Assay||0.25 - 1500 ng/µL|
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