RNA Assay Instructions for Thermo Fisher Qubit™ Fluorometers

Technical Note 201

DeNovix RNA Assay Instructions for Thermo Fisher Qubit™ Fluorometers

Introduction

The DeNovix RNA Assay enables the accurate detection of RNA samples with a standard detection range of 5 ng – 1500 ng total mass in 200 µL volumes. This equates to sample concentrations of 250 pg/µL – 1500 ng/µL when using between 1 and 20 µL sample volumes in 200 µL assay volumes. The assay is linear for sample concentrations as high as 1500 ng/µL when adjusting volumes to 1 µL of sample into 199 µL of working reagent.

The DeNovix RNA Assay is available for use on Thermo Fisher Qubit™ 2.0, 3.0 and Qubit™4 fluorometers. The assay parameter files are available here.

Kit Contents

Three assay sizes are available. The volume of components in each kit are sufficient for 1000, 250 and 50 (evaluation size) assays respectively. Kit components are shown in Table 1. Safety data sheets are available at denovix.com/sds.

Table 1: DeNovix RNA Assay Kit Components

Component	1000	250	EVAL
DeNovix RNA Quantitation Dye (200x)	1 mL	250 µL	50 µL
DeNovix RNA Assay Buffer	250 mL	62.5 mL	12.5 mL
100 ng/µL RNA Standard (mammalian cell)	4 x 400 µL	400 µL	0.1 mL
0 ng/µL RNA Standard	2 mL	0.5 mL	0.5 mL

Best Practices

Use properly calibrated pipettes and RNAse-free pipette tips.
Treat all standards and samples identically in terms of incubation times and temperature.
Generate a new standard curve for each assay.
Ensure that sample solution contaminant levels are compatible with the assay.

Installing DeNovix Assays on a Thermo Fisher Qubit™ Fluorometer

For a Thermo Fisher Qubit™ 2.0 fluorometer:

Download the appropriate .qbt file.
Add the .qbt file to the root directory of a USB drive. Ensure that this is the only .qbt file in the root directory.
Note: The USB drive must have total storage capacity less than 2GB to be successfully read by the Thermo Fisher Qubit™ 2.0 fluorometer.
Unplug the Thermo Fisher Qubit™ 2.0 fluorometer, insert the USB, and turn the fluorometer back on by plugging it in.
Follow the prompts on the screen to download the assays.

For Thermo Fisher Qubit™ 3.0 and Qubit™ 4 fluorometers:

Download the available .qbt file.
Add the .qbt file to the root directory of a USB drive.
Insert the USB into a Thermo Fisher Qubit™ 3.0 / 4 fluorometers.
Navigate to Settings, and select Import Assay.
Select the assay and choose the Thermo Fisher Qubit™ folder where the assay will be stored. (It is recommended to store it under RNA).

Once the assay file is uploaded to the Thermo Fisher Qubit™ fluorometer, simply select the DeNovix RNA Assay to measure RNA samples.

Sample Prep

Allow all solutions to equilibrate to room temperature before use. Vortex, then centrifuge vials briefly before opening to minimize reagent loss on the cap.
Prepare working solution by mixing 10 mL of the assay buffer with 50 µL of the dye. Scale volumes as needed to make enough volume to aliquot 190 µL of the mixture for each standard and unknown.
For each standard or unknown sample, add 190 µL of the working solution into a labeled tube. Adjust volume when adding more or less than 10 µL of the unknown sample.
Note: Use thin-walled, clear 0.5 mL PCR tubes for assay measurements (DeNovix cat #TUBE-PCR-0.5-500 or equivalent). Label only the tops of the tubes.
Add 10 µL of the 0 ng/µL, 100 ng/µL standards or 1 – 20 µL of unknown RNA samples to the respective tubes and mix well. Avoid introducing air bubbles when mixing.
Incubate assay tubes at room temperature for 5 minutes. Protect from light.

Sample Measurements

Select the DeNovix RNA Assay on a Thermo Fisher Qubit™ Fluorometer.
Note: On a Thermo Fisher Qubit™ 2.0 fluorometer, the assay is added in a folder labeled DeNovix.
Select the Read Standard option.
Insert the 0 ng/µL RNA standard tube, lower the lid and tap the Read Standard button.
Insert the 100 ng/µL RNA standard tube, lower the lid and tap the Read Standard button
After measuring the two standards, proceed to measure samples by tapping the Read Samples button.

Measurement Data for Thermo Fisher Qubit™ 2.0 Fluorometer

Three replicates of RNA samples were measured on a Thermo Fisher Qubit™ 2.0 fluorometer and a DeNovix DS-11 FX Fluorometer using the DeNovix RNA Assay. Each sample was prepared by adding between 1-20 µL stock to 199-180 µL working solution respectively, depending on the total mass limits of the assay. The data is presented in graphical and tabular form across the assay dynamic range below.

Table 2: The data presented demonstrate the measurement range of the DeNovix RNA Fluorescence Assay measured on a DeNovix DS-11 FX Fluorometer and Thermo Fisher Qubit™ 2.0 fluorometer.

Expected RNA	Thermo Fisher Qubit™ 2.0 Fluorometer		DeNovix DS-11 FX Fluorometer
ng/µL	ng/µL	StDev	ng/µL	StDev
0.5	0.58	0.005	0.48	0.014
1	1.00	0.006	0.83	0.006
2.5	2.60	0.015	2.39	0.024
5	4.60	0.125	4.49	0.035
10	10.63	0.058	11.18	0.130
25	25.23	0.115	26.89	0.583
50	48.27	0.153	52.05	0.230
100	98.50	1.249	98.34	0.177
1000	970.33	1.248	980.25	1.809
1500	Measured Out of Range		1530.39	22.029

rna assay performance QB2 — Figure 1: DeNovix RNA Fluorescence Assay results measured on a DeNovix DS-11 FX and Thermo Fisher Qubit™ 2.0 fluorometer.

Measurement Data for Thermo Fisher Qubit™ 3.0 / 4 Fluorometers

Three replicates of RNA samples were measured on Thermo Fisher Qubit™ 3.0. and Qubit™ 4 fluorometers and a DeNovix DS-11 FX Fluorometer using the DeNovix RNA Assay. The data is presented in graphical and tabular form across the assay dynamic range below.

Expected RNA	Thermo Fisher Qubit™ 3.0 Fluorometer		Thermo Fisher Qubit™ 4 Fluorometer		DeNovix DS-11 FX Fluorometer
ng/µL	ng/µL	StDev	ng/µL	StDev	ng/µL	StDev
0.5	0.61	0.007	0.60	0.014	0.48	0.014
1	1.06	0.020	1.09	0.031	0.83	0.006
2.5	2.95	0.020	2.88	0.015	2.39	0.024
5	5.51	0.025	5.30	0.147	4.49	0.035
10	12.87	0.153	12.67	0.058	11.18	0.130
25	30.53	0.577	29.83	0.586	26.89	0.583
50	56.73	0.289	55.03	0.462	52.05	0.230
100	99.77	0.252	97.30	1.931	98.34	0.177
1000	997.33	0.305	973.33	0.208	980.25	1.809
1500	Measured Out of Range		Measured Out of Range		1530.39	22.029

rna assay performance QB3and4 — Figure 2: DeNovix RNA Fluorescence Assay measured results measured on a DeNovix DS-11 FX and Thermo Fisher Qubit™ 3.0 and 4 fluorometers.

Summary

The DeNovix RNA Assay enables specific, highly sensitive RNA quantitation across a wide dynamic range. The assay is available for use on a Thermo Fisher Qubit™ fluorometer through the simple application of downloadable assay files.

Qubit™ is a registered trademark of Thermo Fisher Scientific and its subsidiaries and is used for identification and references purposes only. DeNovix, DeNovix products and this website are not endorsed by Thermo Fisher Scientific.

28-MAR-2024

TN 201 DeNovix RNA Assay Instructions for Thermo Fisher Qubit™ Fluorometers