Portelite™ Fluorimetric RNA Quantitation Kit*Optimized for Cytocite™ and Qubit™ Fluorometers*

1. Prepare StrandBrite™ RNA working solution
2. Add 190 µL StrandBrite™ RNA working solution into each 0.2 mL PCR tube
3. Add 10 µL RNA Standards or test samples into each tube
4. Incubate at room temperature for 2 minutes
5. Monitor fluorescence with CytoCite™ or Qubit™ fluorometer

Bring all the kit components at room temperature before starting the experiment.

Make a 200-fold dilution of StrandBrite™ Green (Component A) in Assay Buffer (Component B). For example, to prepare enough working solution for 8 samples, add 5 µL of StrandBrite™ Green (Component A) into 1 mL of Assay Buffer (Component B).

Note: Protect the working solution from light by covering it with foil or placing it in the dark. 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 be a range from 1~20 µL depending on the estimate concentration of RNA sample. The recommend sample volume is 10 µL with the RNA concentration in 0.01~10 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 on10 µL sample volume with the RNA concentration in 0.01~10 ng/µL range.

1. Add 190 µL StrandBrite™ Green working solution into each CytoCite™ sample tube (#CCT100) or equivalent 0.2 mL PCR tube.

   Note: Use thin-wall, polypropylene, clear 0.2 mL PCR tubes such as#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 CytoCite™ or Quibit™ and monitor the fluorescence with green fluorescence channel. Follow the procedure appropriate for CytoCite™ Fluorometer. For instructions, see: https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer

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 1/3 serial dilution with Assay Buffer to get 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0 ng/µL RNA standard dilutions using RNA Standard #2.
2. Add 190 µL StrandBrite™ Green 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 CytoCite™ and monitor the fluorescence with green fluorescence channel.