Three assay sizes are available. The volume of components in each kit are sufficient for 1000, 250 or 50 (evaluation size) assays respectively. Kit components are shown in the table below.Table 1. Kit sizes and the amount of each component contained within each kit.
|DeNovix RNA Assay Quantitation Dye (200x)||1 mL||250 µL||50 µL|
|DeNovix RNA Assay Buffer||250 mL||62.5 mL||12.5 mL|
|RNA Standard, 100 ng/µL||4 x 400 µL||1 x 400 µL||0.1 mL|
|RNA Standard, 0 ng/µL||2 mL||0.5 mL||0.5 mL|
The spectral properties of the dye are excitation/emission of 634/671 nm in the presence of RNA. The kit is compatible with fluorescence microplate readers and fluorometers with the appropriate excitation sources and emission detectors. Specific instructions using the 2 point standard assay with DeNovix fluorometer are included in Technical Note 199.
Mammalian cell RNA is provided as the reference standard. It may be preferable to use an alternative RNA standard more similar (i.e similar size, linear vs. non-linear i.e. tRNA and rRNA) to the unknown samples of interest. For bacterial RNA, consider using a species-specific standard as the strand construction varies widely depending on the species.
Although many instruments including DeNovix DS-11 Series fluorometers offer the option to use previously saved values, it is recommended that a new standard curve be generated at the time of the assay for optimal results.
Assay Linearity and Detection Limits
Fluorescent quantification specifications are often expressed in a variety of conventions. The full detection range (including the extended range) of this assay can be expressed in the following specifications:Table 2. Tolerance of the assay in total mass added to each tube and the corresponding concentration from the sample.
|Absolute mass per assay tube||5 ng to 1500 ng per 200 µL|
|Concentration in sample stock tube||0.25 ng/µL to
The kit is stable for at least 6 months from ship date when stored as recommended.
Table 3. Storage conditions for each component in the DeNovix RNA assay kit.
|Component||Protect from Light||Temperature|
|DeNovix RNA Assay Dye (200x)*||Yes||4°C - Room Temperature|
|DeNovix RNA Assay Buffer||Optional||4°C - Room Temperature|
|RNA Standard, 0 ng/µL||No||4°C - Room Temperature|
|RNA Standard, 100 ng/µL||Yes||-80°C|
*Note: The DeNovix RNA Assay Dye is provided in DMSO, which may freeze if stored at 4°C.
It is important to pay careful attention to pipetting accuracy and overall sample handling techniques when preparing samples for the RNA Fluorescence Quantification Assay.
Preparing diluted standards is not required when using the optimized preconfigured 2 point assay option in the DeNovix FX or QFX software. For the DeNovix User Defined Standards option or for use on microplate readers, prepare RNA standards by serial dilution of the 100 ng/µL standard provided in DEPC water.Table 4. Constructing a multi-point standard curve from the RNA standard provided.
|100 ng/µL||Undiluted stock tube||None|
|50 ng/µL||20 µL of 100 ng/µL standard||20 µL|
|25 ng/µL||10 µL of 100 ng/µL standard||30 µL|
|10 ng/µL||5 µL of 100 ng/µL standard||45 µL|
|2 ng/µL||10 µL of 10 ng/µL standard||40 µL|
|0.5 ng/µL||10 µL of 2 ng/µL standard||30 µL|
|0 ng/µL||None||100 µL|
Sample concentrations are automatically calculated when using a DeNovix DS-11 FX or QFX fluorometer.
For all other instruments, follow the instructions below:
Table 5. The tolerance of the dye in the presence of common solvents while maintaining linearity.
|Compound||Final maximum concentration in assay (200 µL)|
|Ammonium Acetate||5 mM|
|Sodium Chloride||50 mM|
DeNovix dsDNA Quantification Assays
In addition the DeNovix RNA Assay, DeNovix also offers a range of dsDNA fluorescence quantification assays. The range of assays offered is indicated in Table 6.
Table 6: DeNovix dsDNA AssaysAssay Detection Ranges
|DeNovix dsDNA Assay||Range|
|Broad Range||0.1 – 2000 ng/µL|
|High Sensitivity||10 pg/µL – 250 ng/µL|
|Ultra High Sensitivity||0.5 – 300 pg/µL|
Revised 02 Feb 2021