MyQubit microRNA Assay
MicroRNAs (miRNAs) are small, highly conserved RNA molecules that, like small interfering RNAs (siRNAs), modulate the expression of mRNA molecules by base-pairing with them and preventing expression through a variety of mechanisms. They have been shown to act as key regulators of development, cell proliferation, differentiation, and the cell cycle. There are several other classes of small non-coding RNAs as well, including small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and Piwi-interacting RNA (piRNA). These varied species of RNA have tremendous impact on biological processes, yet most traditional RNA isolation methods based on using glass-fiber filers were developed to recover only mRNA and are not very efficient at recovering these smaller RNAs.
Life Technologies offers several kits for the quantitative recovery of small RNAs from a variety of sample types.
The MyQubit miRNA assay for use with the Qubit® 2.0 Fluorometer makes the quantification of miRNA easy and accurate. The miRNA assay described here utilizes a combination of existing Life Technologies reagents, common buffers, and the MyQubit miRNA assay file. This assay can now be permanently uploaded to your Qubit® 2.0 Fluorometer following the guidelines below.
Download the MyQubit miRNA assay file
The assay is based on the microRNA reagent, which exhibits a large increase in fluorescence upon binding to nucleic acids. Although the reagent is not exclusively selective for miRNA, we have been able to reproducibly quantify miRNA in pure samples at levels as low as 0.5 ng in the assay tube following the protocol below. The MyQubit miRNA assay has a core dynamic range of 5– 500 ng/mL miRNA in the assay tube (Figure 1). Because the assay tolerates 1–20 μL of sample in an assay volume of 200 μL, accurate results can be obtained for initial sample concentrations from 50 –100 μg/mL.
Figure 1. Dynamic range of the MyQubit miRNA assay. (A) This plot shows the line corresponding to the curve-fitting algorithm used to calculate concentration in the MyQubit miRNA assay. For reference, the positions of the standards and a set of experimental data points are shown superimposed onto the line, demonstrating that the curve-fitting algorithm gives accurate values for quantitation. (B) The assay has the same attributes as the existing Qubit® assays.
In addition to this assay for quantitating miRNA, the MyQubit firmware allows you to create other assays for your Qubit® 2.0 Fluorometer. All new Qubit® 2.0 instruments are now pre-loaded with the MyQubit firmware, and existing owners can download the firmware. Since the instrument is operated by simple components, creating additional applications is as straightforward as matching spectral compatibility with the LEDs and emission filters. Our Qubit® web page also provides detailed instructions and templates for creating new MyQubit assays. New assays can be based on existing Life Technologies reagents or assays, or can be novel ideas.
Before you begin
It is important that your Qubit® 2.0 Fluorometer be equipped with version 3.10 or later of Qubit® firmware in order to upload new assays using the MyQubit function.
- Quant-iT™ microRNA reagent
- Buffer, 10 mM Tris, 1 mM EDTA (pH 7.5); miRNA Std. 1
- Buffer, 10 mM Tris, 1 mM EDTA (pH 7.5) + 0.01% CHAPS (w/v)
- Silencer® Select GAPDH Positive Control siRNA; miRNA Std. 2
- Qubit® 2.0 Fluorometer
- MyQubit miRNA assay file
- USB drive clear of other .qbt files
- Qubit® Assay Tubes or Axygen PCR-05-C tubes
- Plastic tubes for preparing buffers and dilutions of standards and samples.