MAN0007535 | MP99002 Revision 1.0For Research Use Only. Not for use in diagnostic procedures.

MyQubit microRNA Assay

Introduction

MicroRNAs (miRNAs) are small, highly conserved RNA molecules that, like small interfering RNAs (siRNAs), modulate the expression of their cognate mRNA molecules by base-pairing with them and preventing expression through a variety of mechanisms. miRNAs have been shown to act as key regulators of development, cell proliferation, differentiation, and the cell cycle.

The MyQubit miRNA Assay for use with the Qubit® 2.0 Fluorometer allows easy and accurate quantification of miRNA using a combination of existing Life Technologies’ reagents, common buffers, and the MyQubit miRNA Assay file. The MyQubit miRNA Assay file can be downloaded from www.lifetechnologies.com/qubit and then permanently uploaded to your Qubit® 2.0 Fluorometer following the guidelines described below.

The MyQubit miRNA Assay is based on the Quant-iT™ OliGreen® ssDNA 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 ng/mL–100 μg/mL.

Figure 1 (A) The plot showing the line corresponding to the curve-fitting algorithm (a Modified Hill plot) used to calculate concentration in the MyQubit miRNA Assay. For reference, the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line, demonstrating that the curve-fitting algorithm gives accurate values for quantitation. (B) The assay has the same look and feel of the existing Qubit® assays.

A B

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Curve-Fitting Algorithm

StandardsRaw Data

0 100 200 300 400 500 600

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In addition to the MyQubit miRNA Assay, the MyQubit firmware allows you to create other assays for your Qubit® 2.0 Fluorometer. All new Qubit® 2.0 instruments are pre-loaded with the MyQubit firmware, which can also be downloaded from www.lifetechnologies.com/qubit for installation into existing instruments. Using the MyQubit firmware, additional assays can be created based on existing Life Technologies reagents and assays or on completely novel ideas. Since the instrument is operated by simple commands, creating additional applications can be as straightforward as matching spectral compatibility with the LEDs and emission filters. Detailed instructions and templates for creating new MyQubit assays are also provided on the Qubit® Fluorometric Quantitation website (www.lifetechnologies.com/qubit).

Before You Begin

Firmware Requirements Your Qubit® 2.0 Fluorometer must have V3.10 firmware or later installed for you to be able to upload new assays using the MyQubit function. Before proceeding, make sure that your Qubit® 2.0 Fluorometer has been upgraded to V3.10 firmware or later.

Materials Required • Quant-iT™ OliGreen® ssDNA reagent (Cat. no. O7582)*• 20X TE Buffer (pH 7.5)**; miRNA Std. 1 (Cat. no. T11493)• 20X TE Buffer (pH 7.5) + 0.01% CHAPS (w/v) (e.g., ZOOM® CHAPS, Cat. no.

ZC10003)• Silencer® Select GAPDH Positive Control siRNA (Hs, Mm, Rn); miRNA Std. 2

(Cat. no. 4390849)• Qubit® 2.0 Fluorometer (Cat. no. Q32866)• MyQubit miRNA Assay file (microRNA assay.qbt), available for downloading at

www.lifetechnologies.com/qubit• USB drive clear of other .qbt files• Qubit® Assay Tubes (Cat. no. Q32856) or Axygen PCR-05-C tubes (VWR part no.

10011-830)• Plastic tubes for preparing buffers and dilutions of standards/samples

* The amount of Quant-iT™ OliGreen® ssDNA reagent (O7582) indicated above provides enough material for 1000 miRNA assays using the Qubit® 2.0 Fluorometer and following the protocol described below.

** 10 mM Tris, 1 mM EDTA (pH 7.5).

Downloading the .qbt File from the Web Download the MyQubit microRNA assay file (microRNA assay.qbt) from

www.lifetechnologies.com/qubit and save it directly to your PC. Then, transfer the file from your computer to the root directory of your USB drive. Ensure that you only have a single .qbt file on your USB drive before uploading it to the Qubit® 2.0 Fluorometer.

Note: Downloading a .qbt file from the web directly to your USB drive may result in unexpected behavior.

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Uploading the .qbt File to the Qubit® 2.0 Fluorometer

1.1 Make sure there is only one .qbt file on your USB drive.

1.2 With your Qubit® 2.0 Fluorometer unplugged, insert the USB drive containing the MyQubit miRNA Assay file (microRNA assay.qbt) into the USB port on the instrument.

1.3 Plug the Qubit® 2.0 Fluorometer back in to power it on. The instrument will display the following message: “miRNA assay.qbt file detected. Do you wish to upload?” Click Yes to proceed with the upload, which will take ~2 seconds.

1.4 Once the upload is complete, you will be directed to a new Home Screen displaying a new button called “miRNA”, which indicates that the MyQubit microRNA assay is permanently uploaded to the instrument (Figure 2). You do not need the USB drive to access the assay. Functionality of the pre-existing assays is not affected in any way.

Figure 2 Upload of the MyQubit miRNA Assay. The Qubit® 2.0 Fluorometer will recognize the miRNA assay.qbt file and guide you through the upload process.

Material Storage and Handling • Quant-iT™ OliGreen® ssDNA reagent should be stored at room temperature, protected from light.

• Buffers may be stored at room temperature for short periods of time (weeks); for long-term storage, these components should be stored at 4°C.

• Silencer® Select siRNA should be stored at < –20°C until use. Once reconstituted, the siRNA (and subsequent dilutions) should be stored at 4°C.

• Refer to the detailed product information provided with the materials for additional storage and handling information, including disposal.

Critical Assay Parameters

Assay Temperature The MyQubit miRNA Assay is designed to be performed at room temperature and delivers optimal performance when all solutions are at room temperature (22–28°C). Temperature fluctuations may influence the accuracy of the assay. To minimize temperature fluctuations, store the Quant-iT™ OliGreen® ssDNA reagent and assay buffer at room temperature and insert all assay tubes into the Qubit® 2.0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence; the Qubit® 2.0 Fluorometer can raise the temperature of the assay solution, even over a period of a few minutes. Do not hold the assay tubes in your hand before reading, as this warms the solution and may result in a low reading.

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Incubation Time To allow the MyQubit miRNA Assay to reach optimal fluorescence, incubate the tubes for 2 minutes after mixing the sample or standard with the working solution. After this incubation period, the fluorescence signal is stable for at least 3 hours at room temperature.

Calibrating the Qubit® 2.0 Fluorometer For each assay, you have the choice to run a new calibration or to use the values

from the previous calibration. As you first start using the instrument, perform a new calibration each time. As you become familiar with the assays, the instrument, your pipetting accuracy, and significant temperature fluctuations within your laboratory, determine the level of comfort you have using the calibration data stored from the last time the assay was calibrated. Remember that the fluorescence signal in the tubes containing standards and the samples is stable for at least 3 hours when stored at room temperature. For best results, perform a new calibration each time a new working solution is prepared.

Calculating the Concentration of Your Sample The Qubit® 2.0 Fluorometer gives values for the MyQubit miRNA Assay in ng/mL. This

value corresponds to the concentration after your sample was diluted into the assay tube. To calculate the concentration of your starting sample, use the following equation:

Concentration of your sample = QF value ×200

x

where QF value is the value given by the Qubit® 2.0 Fluorometer, and x is the volume of sample in microliters added to the assay tube.

Dilution Calculator The Dilution Calculator feature of the Qubit® 2.0 Fluorometer calculates the concentration of your original sample based on the volume of sample in the assay tube.

To have the Qubit® 2.0 Fluorometer perform this calculation for you, press Calculate Stock Conc. after a sample measurement is completed. Using the volume scroll wheel, select the volume of your original sample that you added to the assay tube. When you stop scrolling, the Qubit® 2.0 Fluorometer calculates the original sample concentration based on the measured assay concentration.

To save the data from your calculation to the Qubit® 2.0 Fluorometer, press Save on the Dilution Calculator screen. The last calculated value of your measurement is saved to the instrument with a date and time stamp.

The Dilution Calculator generates a concentration for your original sample with the same units as the value in the assay tube given by the Qubit® 2.0 Fluorometer. The units displayed in this calculation cannot be changed on the instrument for assays accessed using MyQubit.

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Experimental Procedure

Preparing Buffers and Reagents

2.1 Allow all reagents to come to room temperature.

2.2 Quant-iT™ OliGreen® ssDNA reagent comes as a 200X concentrate in DMSO. The stock solution is ready-to-use. No intermediate dilutions are required.

2.3 The buffer used to prepare standard dilutions is 1X TE Buffer (10 mM Tris, 1 mM EDTA, pH 7.5), which may be prepared by diluting 20X TE Buffer (Cat. no. T11493) in E-pure water. This buffer solution also acts as Standard #1 in the calibration of the miRNA assay. 1 mL solution of 1X TE Buffer provides enough material for 100 assay calibrations.

2.4 Prepare 1X TE Buffer + 0.01% CHAPS (w/v), which serves as the assay buffer. All dye working solutions should be prepared in TE Buffer + 0.01% CHAPS (w/v). For convenience, you may prepare an aqueous stock solution of 1% CHAPS (w/v) and dilute to 0.01% (w/v) accordingly.

A solution of 200 mL buffer (20 ng of CHAPS in 200 mLof 1X TE buffer) is sufficient for 1000 assays, following the standard MyQubit miRNA Assay protocol described below.

In the presence of CHAPS, dye working solution is stable for at least 3 hours at room temperature.

2.5 Prepare a 100 ng/μL Silencer® Select siRNA solution by adding 650 μL of 1X TE Buffer only (no CHAPS) to the Silencer® Select siRNA (5 nmol, or 65 μg).

2.6 Prepare a 10 ng/μL Silencer® Select siRNA stock solution by diluting the 100 ng/μL siRNA solution 10-fold in 1X TE Buffer only (no CHAPS) (add 100 μL of 100 ng/μL siRNA solution solution to 900 μL of 1X TE Buffer). This solution is Standard #2 in the calibration of the Qubit® miRNA Assay. 1 mL of the siRNA stock solution provides enough material for 100 assay calibrations.

Performing the MyQubit miRNA Assay The protocol below describes the MyQubit miRNA Assay in a total volume of 200 μL

per microplate well (see Table 1). The final concentrations of assay standards and samples have been adjusted for the additional volume based on the dilution scheme outlined in the protocol below.

Refer to the Qubit® 2.0 Fluorometer User Guide, available for downloading at www.lifetechnologies.com/qubit, for detailed instructions on instrument use.

3.1 Set up two Assay Tubes for the standards and one for each user sample. Use only thin-walled, clear 0.5 mL PCR tubes. Acceptable tubes include Qubit® assay tubes (set of 500, Cat. no. Q32856) or Axygen PCR-05-C tubes (VWR, part no. 10011-830).

3.2 Prepare 200 μL of dye working solution for each standard and sample by diluting the Quant-iT™ OliGreen® ssDNA reagent 1:200 in 1X TE Buffer + 0.01% CHAPS (w/v). For example, if you are assaying 5 samples, prepare 1 mL of dye working solution for the samples and 400 μL for the two standards, for a total of 1.4 mL of dye working solution.

3.3 Prepare calibration standards by combining 10 μL of Standard #1 (1X TE Buffer only) with 190 μL of dye working solution, and 10 μL Standard #2 (10 ng/μL of Silencer® Select siRNA, from Step 2.6 above) with 190 μL of dye working solution.

3.4 Prepare samples by combining 1–20 μL of sample with 180–199 μL of dye working solution.

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3.5 Vortex all of the tubes for 2–3 seconds and incubate at room temperature for 2 minutes.

3.6 Calibrate the assay and read samples by selecting “miRNA” from the Home Screen and following the on-screen instructions.

3.7 Optional: Using the Dilution Calculator function of the Qubit® 2.0 Fluorometer, determine the stock concentration of your original sample.

Product List Current prices may be obtained from our website or from our Customer Service Department.

Cat. no. Product Name Unit SizeQ32866 Qubit® 2.0 Fluorometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . eachO7582 Quant-iT™ OliGreen® ssDNA reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each4390849 Silencer® Select GAPDH Positive Control siRNA (Hs, Mm, Rn) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . eachT11493 20X TE Buffer, RNAse-free . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 mLZC10003 ZOOM® CHAPS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 gQ32856 Qubit® Assay Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 set

Table 1 Volume of reagents used in the MyQubit miRNA Assay

Standard Assay Tube

User Sample Assay Tube

Volume of Dye Working Solution (from Step 3.2) 190 μL 180–199 μLVolume of Standard 10 μL —Volume of User Sample — 1–20 μLTotal volume in each Assay Tube 200 μL 200 μL

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23 January 2013