Portelite™ Fluorimetric RNA Quantification Kit *20-1000 ng Broad Range*

1. Add StrandBrite™ Red RNA BR reagent working solution (200 µL).
2. Add test samples (10 µL).
3. Incubate at room temperature for 5 minutes.
4. Monitor fluorescence intensity in Qubit™ fluorometer using red filter.

Important: The following protocol is provided as an example for quantifying total RNA with StrandBrite™ Red RNA BR. Warm all the components to room temperature before opening. No data are available for the mutagenicity or toxicity of StrandBrite™ Red RNA BR stain. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.

Add 2.5 μL StrandBrite™ Red RNA BR (Component A) into 1 mL of RNA Assay Buffer (Component B) and mix well. Protect the working solution from light by covering it with foil or placing it in the dark.

Note: 1 mL of working solution is enough for 5 tests.

Note: We recommend preparing this solution in a plastic container rather than glass, as the dye may adsorb to glass surfaces. For best results, this solution should be used within a few hours of its preparation.

The acceptable sample volume could range from 1~20 µL depending on the estimated concentration of RNA sample. The recommended sample volume is 10 µL with the RNA concentration in 2~100 ng/µL range. If other sample volume is being used, please adjust the dilution factor in the concentration calculations.

The following protocol is generated based on 10 µL sample volume with the RNA concentration in 2-100 ng/µL range.

1. Add 200 µL StrandBrite™ Red RNA BR working solution into each CytoCite™ sample tube (AAT cat# CCT100) or equivalent 0.2 mL PCR tube.
   Note: Use thin-wall, polypropylene, clear 0.2 mL PCR tubes such as AAT cat# CCT100.
   
2. Add 10 µL RNA standard #1 and #2 or test samples into each tube, and then mix by vortexing 2~3 seconds.
3. Incubate all tubes at room temperature for 2 minutes.
4. Insert the samples into Qubit™ and monitor the fluorescence with red fluorescence channel.

For StrandBrite™ assays, you have the choice to make a calibration curve with the RNA standards. Here is a brief protocol to generate a customized RNA standard curve:

1. Perform dilution of 2 mg/mL RNA standard (Component D) to 100, 80, 60, 40, 20, 10, 5, 2 ng/uL in RNA Assay Buffer (Component B).
2. Add 200 µL StrandBrite™ Red DNA BR working solution into each tube.
3. Add 10 µL RNA standards or test samples into each tube, and then mix by vortexing 2-3 seconds.
4. Incubate the reaction at room temperature for 2 minutes.
5. Insert the samples into Qubit™ and monitor the fluorescence with red fluorescence channel.