Modulus™ Single Tube Fluorometer Method for

Quant-iT™ RNA

1. INTRODUCTION

The Modulus™ Fluorometer from Turner BioSystems in combination with the Quant-iT™ RNA Assay kit from Molecular Probes provides an accurate method for quantitation of RNA in small volumes (100 µL). The Quant-iT RNA assay is highly selective for RNA over double-stranded DNA. When analyzed in combination with the Modulus Fluorometer and the Red Fluorescence Optical Kit, the signal is linear from 0.8-50 ng RNA in 100 µL final volume (Figure 1). The Quant-iT RNA Assay tolerates salts, solvents, detergents, protein and other common contaminants.

Figure 1. rRNA and Quant-iT RNA analyzed using the Modulus and the Red Fluorescence Optical Kit. 5 µL of each standard was added to 100 µL of Quant-iT RNA reagent. 100 µL of this mixture was susequently transferred to a minicell cuvette and Modulus measured under the Raw Fluorescence Mode of each dilution.

2. MATERIALS REQUIRED

• Modulus Fluorometer (P/N 9200-000 or 9200-002)
• Red Fluorescence Optical Kit (P/N 9200-043)
• Quant-iT RNA Assay Kit (Molecular Probes, Q33140)
• Minicell Cuvettes (P/N 7000-950) and Minicell Adaptor (P/N 9200-928)

3. EXPERIMENTAL PROTOCOL

3.1 Reagent Preparation

NOTE: Handling, storage and use of the reagent should be performed in accordance with the product information sheet supplied by Molecular Probes, Inc.

The Quant-iT RNA Reagent is supplied as a 1 mL concentrated dye solution in anhydrous dimethylsulfoxide (DMSO). On the day of the experiment, equilibrate kit contents to room temperature. Prepare a working solution of the Quant-iT RNA Reagent by making a 1:200 dilution of the concentrated dye solution in Quant-iT RNA buffer. Prepare this solution in a plastic container as the reagent may absorb to glass surfaces. Protect the working solution from light by covering it with foil or placing it in the dark.

NOTE: For best results, use this solution within 3 hours of its preparation.

3.2 Instrument Set-Up

3.2.1 Power OFF the Modulus. Insert the Red Fluorescence Optical Kit and Minicell Adaptor according to the Operating Manual.

3.2.2 Turn ON the Modulus. Allow a 5-minute warm up period before calibration.

3.3 Calibration

3.3.1 Add 5 µL of each standard to a microcentrifuge tube containing 100 µL of the Quant-iT RNA reagent working solution. Mix by inversion.

3.3.2 Transfer 105 µL of each standard to a minicell cuvette.

3.3.3 Calibrate the Modulus with as many as 5 of the standards. Choose "ng/µL" for the unit of measure. Use the 0 ng/µL standard for the blank solution. To optimize performance and accuracy, choose the 5 standards that are closest in range to a typical sample. Enter the standards in order of increasing concentration.

3.3.4 Save the calibration for future use (optional).

3.4 Sample Analysis

3.4.1 Add 5 µL of each sample to a microcentrifuge tube containing 100 µL of the Quant-iT RNA Reagent working solution. Mix by inversion.

3.4.2 Transfer 105 µL of each sample to a minicell cuvette.

3.4.3 Read each sample. The concentration of the sample in ng/µL appears on the touchscreen.

NOTE: It is not necessary to run a standard curve after calibration. To check the linearity of the calibration, re-read the standards as samples.

4. PATENTS AND TRADEMARKS

Quant-iT is a registered trade--mark of Molecular Probes,Inc.

Modulus is a trademark of Turner BioSystems, Inc.

5. ABOUT MOLECULAR PROBES, INC.

Orders for Molecular Probes' products may be placed by:

Phone: (541) 465-8338 or
Toll-Free: (800) 438-2209 (U.S. and Canada)

Fax: (541) 344-6504 or
Toll-Free: (800) 438-0228 (U.S. and Canada)
E-mail: order@probes.com

Mailing Address:
Molecular Probes, Inc.
PO Box 22010
Eugene, OR 97402-0414 USA

Information on the scientific and technical back-ground of Molecular Probes' products is available from the Technical Assistance Department:

Phone: (541) 465-8353
Fax: (541) 465-4593
E-mail: tech@probes.com
Internet: http://www.probes.com

6. ABOUT TURNER BIOSYSTEMS, INC.

Orders for Turner BioSystems' products may be placed by:

Phone: (408) 636-2400
Toll Free: (888) 636-2401 or
Fax: (408) 737-7919

Contact us via our contact form
Internet: www.turnerbiosystems.com

Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085

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