Promega E3310 QuantiFluor RNA System Instruction Manual

Promega E3310 QuantiFluor RNA System

Product Information

• Specifications
  • Product: 25ml 20X TE Buffer (pH 7.5)
  • Size: 1ml
  • CAT. #: E3310
• Product Components and Storage Conditions
  • The product includes a 25ml 20X TE Buffer (pH 7.5) stored in a 1ml size container with CAT. # E3310.

Product Usage Instructions

• Protocol for Quantitating RNA in a Single Tube Using the QuantusTM Fluorometer
  • Step 1: Add prepared blank, high or low standard, and unknown samples to 0.5ml tubes. Mix and incubate for 5 minutes.
  • Step 2: Measure fluorescence.
  • Note: If the QuantusTM Fluorometer was previously calibrated, you may skip certain steps.
• Steps for Preparation:
  • Prepare 1X TE buffer by diluting the 20X TE Buffer 20-fold with nuclease-free water.
  • Prepare a working solution as per instructions.
  • Prepare RNA standard sample.
• Measurement Process:
  • Incubate samples at room temperature for 5 minutes, protected from light.
  • Select the RNA protocol on the QuantusTM Fluorometer.
  • If needed, calibrate the Fluorometer and read blank and standard samples.
  • Measure the fluorescence of the unknown sample using the QuantusTM Fluorometer.
• Protocol for Quantitating RNA in Multiwell Plates
  • Detailed steps for quantitating RNA in multiwell plates are provided with specific instructions for preparing standard curves and samples.

FAQs

• Q: What is the recommended storage condition for the product?
  • A: The product should be stored according to the instructions provided on the packaging or technical manual to maintain its integrity and performance.
• Q: Can I use other buffer solutions instead of the provided TE Buffer?
  • A: It is recommended to use the specified TE Buffer for accurate results. Substituting with other buffers may affect the assay performance.

Description

• The QuantiFluor® RNA System(a) contains a fluorescent RNA-binding dye (492nmEx/540nmEm) that enables sensitive quantitation of small amounts of RNA in purified samples.
• The assay is linear over a range of 0.1–500ng of RNA input (0.1–500ng/μl from 1μl of the original sample).
• The dye-based system provides concentrated QuantiFluor® RNA dye, dilution buffer and RNA standard.
• Simply dilute the dye, add standards and unknown samples, and read on a fluorometer.
• The QuantiFluor® RNA System can be used with any single-tube fluorometer (e.g., Quantus™ Fluorometer [Section 3]) or may be scaled up for use in instruments that can read multiwell plates (e.g., GloMax® Discover System [Section 4]) at the appropriate excitation and emission wavelengths.

Product Components

Product Components and Storage Conditions

This system contains sufficient reagents for 2,000 assays at a 200μl scale. Includes:

• 25ml 20X TE Buffer (pH 7.5)
• 1ml QuantiFluor® RNA Dye
• 100μg RNA Standard, 100μg/ml

Storage Conditions: Store the QuantiFluor® RNA Dye at –30°C to –10°C, protected from light. Store the RNA Standard, 100μg/ml, below –10°C. Store the 20X TE Buffer at –30°C to +30°C. Instructions for handling and disposal of QuantiFluor® RNA Dye are located in Section 7. E.

Protocol for Quantitating

Protocol for Quantitating RNA in a Single Tube Using the Quantus™ Fluorometer

• Materials to Be Supplied by the User
  • nuclease-free water
  • thin-walled 0.5ml PCR tubes (Cat.# E4941 or Axygen Cat.# PCR-05-C)
  • optional: RQ1 RNase-Free DNase (Cat.# M6101) to remove contaminating DNA; Section 7.C

Warm all assay components to room temperature before use. The QuantiFluor® RNA Dye is dissolved in 100% DMSO and frozen at or below 4°C. Before dilution, thaw the dye at room temperature, protected from light. The QuantiFluor® RNA System enables accurate detection of RNA over a broad dynamic range. Quantitating unknown samples requires instrument calibration using a blank and a single standard sample. Prepare a standard sample using the 1.2kb RNA Standard. This standard should be appropriate for the expected range of nucleic acid concentrations for your unknown samples. For samples in the range of 10–500ng/μl, prepare a working solution and standard sample by following instructions for the “High Standard Calibration.” For lower-level samples in the range of 0.1–10ng/μl, prepare a working solution and a standard sample according to the “Low Standard Calibration” instructions. Instructions for use of the Quantus™ Fluorometer can be found in the Quantus™ Fluorometer Operating Manual #TM396 available at: www.promega.com/protocols. We recommend preparing a standard curve that extends above and below the likely concentration range for your unknown samples. In addition, a blank sample containing 1X TE buffer should be used to assess the background level of the assay.
Note: Other single-tube fluorometers can be used with the QuantiFluor® RNA System is capable of measuring the following wavelengths (492nmEx/540nmEm) and calibrated using manufacturer’s instructions.

• Step 1
  • Prepare QuantiFluor® RNA Dye working solution for high- or low concentration measurements, and dispense into 0.5ml tubes, enough for each blank, standard and unknown sample.
• Step 2
  • Add prepared blank, high or low standard and unknown samples to 0.5ml tubes. Mix, and incubate for 5 minutes.
• Step 3
  • Measure fluorescence.

Overview

Figure 1. Overview of the single-tube format quantitation using the QuantiFluor® RNA System.

Note: If the Quantus™ Fluorometer was previously calibrated, you may not need to calibrate it again. Therefore, do not prepare blank and standard samples, and skip Steps 3, 4 and 8.

1. Prepare 1X TE buffer: Dilute the 20X TE Buffer 20-fold with nuclease-free water (not provided). For example, add 1ml of 20X TE Buffer to 19ml of Nuclease-Free Water (Cat.# P1195), and mix.

2. Prepare Working Solution: High Standard Calibration: Dilute the QuantiFluor® RNA Dye 1:400 in 1X TE buffer to make the QuantiFluor® RNA Dye working solution. For example, add 10μl of QuantiFluor® RNA Dye to 3,990μl of 1X TE buffer, and mix thoroughly. Low Standard Calibration: Dilute the QuantiFluor® RNA Dye 1:2,000 in 1X TE buffer to make the QuantiFluor® RNA Dye working solution. For example, add 2μl of QuantiFluor® RNA Dye to 3,998μl of 1X TE buffer, and mix thoroughly. Protect the working solution from light by covering it with foil or placing in the dark.
   Note: The QuantiFluor® Dye working solution is stable for 2–3 hours at 25°C.

3. Prepare Blank Sample: Add 200μl of QuantiFluor® RNA Dye working solution to an empty 0.5ml PCR tube. This will be the blank sample used in Step 8. Protect the tube from light.

4. Prepare RNA Standard Sample: High Standard Calibration: Prepare a 500ng standard by adding 5μl of the provided RNA Standard (100ng/μl) to 200μl of QuantiFluor® RNA Dye working solution in an empty 0.5ml PCR tube. Mix, and protect the tube from light. Low Standard Calibration: Prepare a 10ng standard by first diluting the RNA Standard (100ng/μl) 1:100 in 1X TE buffer (10μl of RNA Standard + 990μl of 1X TE buffer). Next, add 10μl of diluted standard to 200μl of QuantiFluor® RNA Dye working solution in a 0.5ml PCR tube.

5. Protocol for Quantitating RNA in a Single Tube Using the Quantus™ Fluorometer (continued)

6. Prepare Unknown Sample: Add 1–20μl of an unknown sample to 200μl of QuantiFluor® RNA Dye working solution in 0.5ml PCR tubes. For example, add 1μl sample to 200μl QuantiFluor® RNA Dye working solution. Vortex well, and protect the tube from light.

   • Note: See Section 7.D, Tips for Pipetting Small Volumes, if you are pipetting 1μl samples. Alternatively, minimize the pipetting error by using >1μl of the sample.

7. Incubate the prepared samples at room temperature for 5 minutes, protected from light.

8. Select the RNA protocol on the Quantus™ Fluorometer. Depending on which standard calibration you prepared, select “High” or “Low.”

9. If needed, calibrate the Quantus™ Fluorometer by reading the blank (prepared in Step 3) and standard (prepared in Step 4) samples in the Calibration screen, and then select “Save”.

10. Enter the volume of the unknown sample and desired concentration units.

    • Note: This volume is the amount of sample that is added for the quantitation. For example, if 2μl of the sample was mixed with 200μl of QuantiFluor® RNA Dye working solution, then the volume entered on this screen should be 2μl.

11. Measure the fluorescence of the unknown sample using the Quantus™ Fluorometer. The number displayed represents the concentration of the original sample.

Protocol for Quantitating RNA in Multiwell Plates

• Materials to Be Supplied by the User
  • multiwell plate detection instrument capable of measuring fluorescence (e.g., GloMax® Discover System [Cat.# GM3000])
  • Nuclease-Free Water (Cat.# P1195)
  • black flat-bottom 96-well plates
  • optional: RQ1 RNase-Free DNase (Cat.# M6101) to remove contaminating DNA; Section 7.C

Warm all assay components to room temperature before use. The QuantiFluor® RNA Dye is dissolved in 100% DMSO and frozen at or below 4°C. Before dilution, thaw the dye at room temperature, protected from light. Instructions for use of the GloMax® Discover System can be found in the GloMax® Discover System Operating Manual #TM397, available at: www.promega.com/protocols.

• Step 1
  • Prepare QuantiFluor® RNA Dye working solution for high- or low-concentration measurements and dispense it into a multiwell plate.
• Step 2
  • Add blank, prepared standards and unknown samples to the multiwell plate. Mix, and incubate for 5 minutes.
• Step 3
  • Measure fluorescence.

Figure 2. Overview of multiwell plate quantitation protocol using the QuantiFluor® RNA System.

The QuantiFluor® RNA System can accurately assess RNA concentration over a broad dynamic range. For samples 10–500ng/μl, prepare working solution and a standard curve by following instructions for the “High-Concentration Standard Curve.” For lower concentration samples (0.1–10ng/μl), prepare working solution and a standard curve according to the “Low-Concentration Standard Curve” instructions. The following protocols are examples of how to prepare standard curves of two different concentration ranges. We recommend preparing a standard curve that extends above and below the likely concentration range for your unknown samples. In addition, a blank sample containing 1X TE Buffer should be used to assess the background level of the assay.

1. Prepare 1X TE buffer: Dilute the 20X TE Buffer 20-fold with nuclease-free water (not provided). For example, add 1ml of 20X TE Buffer to 19ml of Nuclease-Free Water (Cat.# P1195), and mix.
2. Prepare Working Solution: Prepare enough QuantiFluor® RNA Dye working solution to quantitate blank, standard and unknown samples. Protect the working solution from light by covering with foil or placing it in the dark. High Standard Curve (for samples 10–500ng/μl): Dilute the QuantiFluor® RNA Dye 1:400 in 1X TE buffer to make the QuantiFluor® RNA Dye working solution. For example, add 10μl of QuantiFluor® RNA Dye to 3,990μl of 1X TE buffer, and mix thoroughly. Low Standard Curve (for samples 0.1–10ng/μl): Dilute the QuantiFluor® RNA Dye 1:2,000 in 1X TE buffer to make the QuantiFluor® RNA Dye working solution. For example, add 2μl of QuantiFluor® RNA Dye to 3,998μl of 1X TE buffer, and mix thoroughly.
   • Note: The QuantiFluor® RNA Dye working solution is stable for 2–3 hours at 25°C.
3. Protocol for Quantitating RNA in Multiwell Plates (continued)
4. Prepare RNA Standard Curve: High-Concentration Standard Curve: The following recommended standards result in 7.8–500ng/well, and are designed for optimal pipetting accuracy, transferring 10μl of standard to each well.
   • a. Prepare seven 1.5ml tubes labeled: 500, 250, 125, 62.5, 31.3, 15.6, 7.8.
   • b. Prepare RNA standards by serially diluting QuantiFluor® RNA Standard (100ng/μl) as shown in Table 1. Take care to not introduce air bubbles.

Table 1. Preparing High-Concentration RNA Standard Curve Samples.

Standard| The volume of RNA Standard| Volume of 1X TE Buffer| Final RNA Concentration (ng/µl)
---|---|---|---
A| 50µl| 50µl| 50
B| 50µl of Standard A| 50µl| 25
C| 50µl of Standard B| 50µl| 12.5
D| 50µl of Standard C| 50µl| 6.25
E| 50µl of Standard D| 50µl| 3.13
F| 50µl of Standard E| 50µl| 1.56
G| 50µl of Standard F| 50µl| 0.78

Low-Concentration Standard Curve: The following recommended standards result in 0.16–10ng/well and are designed for optimal pipetting accuracy, transferring 10μl of standard to each well.

• a. Prepare seven 1.5ml tubes labelled: 10, 5, 2.5, 1.3, 0.63, 0.31, 0.16.
• b. Prepare RNA standards by serially diluting QuantiFluor® RNA Standard (100ng/μl) as shown in Table 2. Take care to not introduce air bubbles.

Table 2. Preparing a Low-Concentration RNA Standard Curve.

**Standard| The volume of RNA Standard| Volume of 1X TE Buffer| Final RNA Concentration (ng/µl)**
---|---|---|---
A| 10µl| 990µl| 1.0
B| 50µl of Standard A| 50µl| 0.5
C| 50µl of Standard B| 50µl| 0.25
D| 50µl of Standard C| 50µl| 0.13
E| 50µl of Standard D| 50µl| 0.063
F| 50µl of Standard E| 50µl| 0.031
G| 50µl of Standard F| 50µl| 0.016

1. Pipet 200μl of QuantiFluor® RNA Dye working solution into each well that is intended for an unknown, blank or standard sample.
2. Dispense 10μl of the high-concentration RNA standards prepared in Table 1 (labelled Standards A–G) to rows A–G of the 96-well plate (Figure 3, Panel A). Alternatively, dispense 10μl of the low-concentration RNA standards prepared in Table 2 (labelled Standards A–G) to rows A–G of the 96-well plate (Figure 3, Panel B). We recommend pipetting duplicates or triplicates of the standards.
3. For the blank, pipet 10μl of 1X TE buffer into row H.
4. Add 1–20μl of unknown sample to the remaining wells. Record the dilution factor that was used for each unknown sample. The dilution factor will be used when calculating the concentration of the unknown sample (Section 5, Step 4).

Protocol for Quantitating RNA in Multiwell Plates (continued)

1. Protocol for Quantitating RNA in Multiwell Plates (continued) Figure 3. Dispense standard dilutions and blank samples in duplicate into Columns 1 and 2 of a multiwell plate. Panel A. High-concentration RNA standard and blank samples. Panel B. Low-concentration RNA standard and blank samples.
2. Mix the plate thoroughly using a plate shaker or by pipetting the contents of each well.
3. Incubate assays for 5 minutes at room temperature, protected from light.
4. Measure fluorescence (492nmEx/540nmEm) using your plate reader. If using the GloMax® Discover System, select the preloaded protocol: “QuantiFluor RNA System.”.
5. Calculate the RNA concentration as follows: Subtract the fluorescence of the blank sample (1X TE buffer) from all of the standard and unknown samples. Use the corrected data from the RNA standards to generate a standard curve of fluorescence versus RNA concentration. Determine the RNA concentration of the sample from the standard curve and multiply the resulting number by the dilution factor, if applicable. Alternatively, copy and paste your raw fluorescence data into our online tool: www.promega.com/resources/tools/quantifluor-dye-systems-data-analysis-workbook.
6. Representative Data Representative data for the QuantiFluor® RNA Dye are shown in Table 3 and Figure 4.

Table 3. Representative Data for the High-Concentration RNA Standard Curve and QuantiFluor® RNA Dye in 96-Well Plate Format.

Representative Data

Figure 4. Representative RNA standard curves in a 96-well-plate format. Panel A. The high-concentration RNA standard curve. Panel B. The low-concentration RNA standard curve. The final concentrations of RNA standard in 96-well plate, 200μl assay format are listed in Table 3.

Example Calculation:
The standard and unknown samples have these average fluorescence values (in RFU):

 |

Unknown Sample

| Standard Samples
---|---|---
0ng| 7.8ng| 15.6ng| 31.3ng| 62.5ng| 125ng| 250ng| 500ng
Fluorescence| 17,775| 944| 2,094| 3,796| 7,795| 16,901| 36,035| 80,326| 152,705

1. Subtract the 1X TE buffer blank (average of blank standards) from all samples:
    |

Unknown Sample

| Standard Samples
---|---|---
0ng| 7.8ng| 15.6ng| 31.3ng| 62.5ng| 125ng| 250ng| 500ng
Fluorescence| 16,831| 0| 1,150| 2,852| 6,851| 15,957| 35,091| 79,382| 151,761
2. Determine the linear regression from the standard curve (Figure 4).

• y = 309.3x – 1730.5

3. Calculate the RNA concentration of the unknown sample in the 200μl assay volume by solving for x in the linear regression equation, where
   • y = 16,831
   • x = (y + 1730.5)/309.3 = 60ng
4. Account for any dilution of the unknown sample. For example, if 1μl of the sample was added per well, the sample concentration is 60ng/μl. If 5μl of the sample was added per well, the sample concentration is 60ng ÷ 5μl = 12ng/μl.

Troubleshooting

For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information is available at: www.promega.com.
E-mail: [email protected].

www.promega.com/resources/tools/quantifluor-dye-systemsdata-analysis- workbook.

Appendix

Instrument Compatibility
Fluorescence measurements can be performed using any fluorescence reader capable of measuring excitation and emission at the appropriate wavelengths. For the QuantiFluor® RNA Dye, measure excitation and emission at 492nm and 540nm, respectively; emission and excitation spectra are shown in Figure 5.

Figure 5. Excitation and emission spectra for the QuantiFluor® RNA Dye.

Interfering Compounds

• Several compounds that are commonly used in nucleic acid preparation or can be found in eluates from nucleic acid purification may affect the QuantiFluor® RNA Dye.
• Table 4 lists compounds that have known effects on RNA quantitation using the QuantiFluor® RNA Dye and the concentrations at which they affect quantitation results.

Table 4. Compounds that Interfere with the QuantiFluor® RNA Dye.

Chemical| Concentration Shown to Affect the QuantiFluor ® RNA Dye 1| Change in QuantiFluor ® RNA Dye Signal
---|---|---
agarose| 0.01%| 16% decrease
ammonium acetate| 20mM| 11% decrease
bovine serum albumin (BSA)| 0.003%| 9% increase
chloroform| 0.4%| 1% decrease
ethanol| 20%| 18% decrease
IgG| 0.02%| 1% decrease
boric acid| 150mM| 8% decrease
formamide| 5%| 3% decrease
5X Transcription Reaction Buffer| 0.25%| 15% decrease
ImProm-II™ 5X Reaction Buffer| 2%| 4% increase
GoScript™ 5X Reaction Buffer| 2%| 6% decrease
sodium chloride| 20mM| 12% decrease
magnesium chloride| 1mM| 6% decrease
calcium chloride| 1mM| 2% decrease
zinc chloride| 1mM| 12% decrease
1,2-propanediol| 20%| 11% increase
cesium chloride| 10mM| 6% decrease
guanidine thiocyanate| 10mM| 7% decrease
sucrose| >500mM| 9% increase
urea| 3M| 23% decrease
phenol| 0.5%| 13% decrease
polyethylene glycol (PEG 8000)| 20%| 1% decrease
sodium acetate| 20mM| 9% decrease
sodium dodecyl sulfate (SDS)| 0.005%| 3% increase
Triton® X-100| 0.5%| 9% decrease
1Compounds were tested at the indicated QuantiFluor® RNA Dye.| concentration with 20ng per well of RNA St| standard and a 1:1,000 dilution of

Sample Considerations

For RNA samples containing a mixture of RNA and DNA, we recommend a DNase treatment using RQ1 RNase-Free DNase (Cat.# M6101) to degrade contaminating DNA and quantitate RNA more specifically. For samples containing significant amounts of DNA, treat the RNA sample before quantitation. Follow the RQ1 RNase-Free DNase Usage Information on the Cat.# M6101 Certificate of Analysis. Below is an example protocol for a final reaction volume of 10μl.

1. Add 1μl of RQ1 RNase-Free DNase 10X Reaction Buffer to each unknown RNA sample.
2. Add 1μl of RQ1 RNase-Free DNase to each sample, and mix gently. Do not vortex.
3. Incubate at 37°C for 30 minutes.
4. Add 1μl of RQ1 DNase Stop Solution to terminate the reaction.
5. Incubate at 65°C for 10 minutes to inactivate the DNase.
6. Proceed to RNA quantitation in Section 4.

Tips for Pipetting Small Volumes

1. Handheld pipettes are high-precision instruments and should be handled carefully. Pipettes also should be routinely checked for accuracy every 6–12 months and recalibrated or repaired by qualified professionals.
2. Use newer model pipettes with more modern “press fit” tips for the most accurate and reproducible results.
3. Basic pipetting guidelines:
   • When resetting a pipette to a new volume, turn the adjustment knob ½ turn higher than the target volume and then rotate back to the target volume.
   • Pipet uses a slow, smooth action.
   • Hold the pipette vertically when drawing in liquid.
   • Only immerse the pipette tip slightly when aspirating the liquid.
   • When dispensing the liquid, hold the pipette vertically but keep the sidewall of the receiving vessel at a 45-degree angle. Pipette against the sidewall or into a liquid that is already present.
4. Use the proper pipette for the measured volume. When pipetting 1μl or 2μl, use a P2 pipettor for accuracy and reproducibility. Using aerosol or barrier tips with a P2 is not recommended.
5. All solutions, standards and unknowns should be at room temperature before pipetting. See the following Nature
   • Methods Application Note for more information: www.nature.com/app_notes/nmeth/2007/071109/full/nmeth1086.html.
   • For a comprehensive guide on pipetting, we recommend the following resource: www.gilson.com/default/guide-to-pipetting.

Handling and Disposal

• QuantiFluor® RNA Dye contains DMSO, which is an irritant and facilitates the entry of organic compounds into tissues.
• Wear gloves, safety glasses and a lab coat, and handle dye with care.
• Because the QuantiFluor® RNA Dye binds to nucleic acid, it should be treated as a potential mutagen.
• Dispose of the QuantiFluor® RNA Dye according to local regulations.

Composition of Buffers and Solutions

• 20X TE Buffer (pH 7.5)
• 0.2M Tris buffer (pH 7.5)
• 20mM EDTA (pH 8.0)

Prepare this solution in nuclease-free water. Adjust pH to 7.5.

Related Products

Summary of Changes
The following changes were made to the 3/24 revision of this document:

1. Updated font and cover image
2. Made minor text edits.
3. Updated patent statements.

(a)U.S. Pat. Nos. 8,598,198 and 9,206,474 and other patents and patents pending. © 2013–2024 Promega Corporation. All Rights Reserved. GloMax and QuantiFluor are registered trademarks of Promega Corporation. NoScript, ImProm-II and Quantus are trademarks of Promega Corporation. Triton is a registered trademark of Union Carbide Chemicals & Plastics Technology Corporation. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. All prices and specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalogue for the most up-to-date information on Promega products.

CONTACT

• Promega Corporation
• 2800 Woods Hollow Road
• Madison, WI 53711-5399 USA
• Toll-Free in USA 800-356-9526
• 608-274-4330
• Fax 608-277-2516 19
• www.promega.com.
• TM377 · Revised 3/24

References

• Advertising Feature: Application Note | Nature Methods
• Promega Corporation
• Protocols
• Protocols
• QuantiFluor® Dye Systems Data Analysis Workbook

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