Application Note

Sensitive RNA fluorescent quantitation with the Quant-iT RiboGreen RNA assay kit

  • Sensitive fluorescent quantitation of RNA down to 100 pg/mL
  • Linear dynamic range spanning four orders of magnitude
  • Easy data acquisition and analysis with preconfigured protocol in SoftMax Pro Software

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Beverly Pappas, PhD | BioResearch Field Applications Scientist | Molecular Devices

Introduction

Accurate quantification of nucleic acid concentration is important for downstream applications including transfection, cloning, PCR, and next generation sequencing (NGS). Often, these applications have specific target nucleic acid concentrations for optimal performance. Inaccurate quantitation can increase variability in downstream assays and affect the quality of results1.

RNA is typically quantitated in microplate readers by measuring the absorbance at 260 nm. However, absorbance-based methods can have disadvantages, including contaminant interference, signal contribution from nonspecific proteins and free nucleotides, and sensitivity limits2. The Quant-iT™ RiboGreen RNA Assay Kit from Thermo Fisher Scientific is more specific for RNA quantitation. The dynamic range of this assay in microplate format, as stated in the product manual, is from 1 ng/mL to 1 μg/mL using two dye concentrations, about 1000-fold more sensitive than absorbance methods3. In this application note, we demonstrate that with the Quant-iT RiboGreen assay, users can reliably measure concentrations as low as 100 pg/mL of RNA with the SpectraMax® iD5 Multi-Mode Microplate Reader, with similar results obtained on the other SpectraMax readers used.

To maximize sensitivity of the assay, the Spectral Optimization Wizard was used for reads obtained with the SpectraMax iD5 and SpectraMax® i3x Multi-Mode Microplate Readers to determine optimal excitation and emission wavelengths. By using the Spectral Optimization Wizard, the dual monochromators in the SpectraMax iD5 and i3x readers can hone in on the optimal wavelength pair to provide the best sensitivity and dynamic range for the assay.

Materials

Methods

Instrument and protocol setup

Prepare the assay

The method outlined below for this assay follows the instructions in the product information sheet for the Quant-iT RiboGreen RNA Reagent and Kit, except the assay volume is proportionately reduced from 2.0 mL to 200 μL to fit a 96-well microplate format.

Read the microplate

Analyze the data

Parameter
SpectraMax iD5/iD3
SpectraMax i3x

SpectraMax

M5/M5e/M4/M3/M2/M2e

SpectraMax Mini
Read mode
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Read type
Endpoint
Endpoint
Endpoint
Endpoint
Wavelengths

Excitation: 480

Emission: 520

Excitation: 480,

Bandwidth 9nm

Emission:520,

Bandwidth 15nm

Excitation: 480

Emission: 525

Emission Cutoff: 515

FL-535 filter cube

Excitation: 485/20

Emission: 535/25

Plate type and read area
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
PMT and optics
PMT gain: Automatic Integration time: 200 ms Read height: optimize for microplate used
PMT gain: N/A Flashes per read: 6 Read Height: Optimize for microplate used
PMT gain: Automatic Flashes per read: 6
PMT gain: Automatic Integration time: 200 ms Read height: optimize for microplate used

***Table 1.*Instrument settings for SpectraMax readers. For SpectraMax iD5, i3x, and Mini readers, the read height setting should be optimized for the microplate used. Note: Additional readers with similar performance are listed.

Standard
Volume (μL) of 1X TE
Volume (μL) of 2 μg/mL RNA Stock
Volume (μL) of 200-fold diluted Quant-iT™ RiboGreen Reagent
Final RNA concentration in Quant-iT™ RiboGreen Assay
High-Range Standard 1
0
500
500
1 μg/mL
High-Range Standard 2
250
250
500
500 ng/mL
High-Range Standard 3
450
50
500
100 ng/mL
High-Range Standard 4
490
10
500
20 ng/mL
High-Range Blank
500
0
500
0

***Table 2.*Protocol for preparing high-range standard curve.

Volume (μL) of 1X TE
Volume (μL) RNA
Working RNA Concentration (ng/mL)
Dilution 1
0
1000 μL of 100 ng/mL RNA
50
Dilution 2
500
500 μL of Dilution 1
25
Dilution 3
800
200 μL of Dilution 2
5
Dilution 4
800
200 μL of Dilution 3
1
Dilution 5
800
200 μL of Dilution 4
0.2
Dilution 6
800
200 μL of Dilution 5
0.04
Dilution 7
800
200 μL of Dilution 6
0.008
Dilution 8
800
200 μL of Dilution 7
0.0016
Dilution 9
600
0
0

***Table 3a.*Protocol for preparing low-range dilutions. Serial RNA dilutions are made using 1X TE and the 100 ng/mL RNA stock solution.

Low-Range Dilution
Volume (μL) of 2000-fold Diluted Quant-iT™ RiboGreen® Reagent
Final RNA Concentration in Quant-iT™ RiboGreen® Assay (ng/mL)
Low-Range Standard 1
500 μL of Dilution 1
500
25
Low-Range Standard 2
500 μL of Dilution 2
500
12.5
Low-Range Standard 3
500 μL of Dilution 3
500
2.5
Low-Range Standard 4
500 μL of Dilution 4
500
0.5
Low-Range Standard 5
500 μL of Dilution 5
500
0.1
Low-Range Standard 6
500 μL of Dilution 6
500
0.020
Low-Range Standard 7
500 μL of Dilution 7
500
0.004
Low-Range Standard 8
500 μL of Dilution 8
500
0.0008
Low-Range Blank
500 μL of Dilution 9
500
0

***Table 3b.*Protocol for preparing low-range standard curve. 2000-fold diluted Quant-iT RiboGreen reagent is added to low-range dilutions from Table 3a to obtain final RNA concentrations.

Results

RNA standards ranging from 100 pg/mL to 1 μg/mL were detected using the Quant-iT RiboGreen assay kit and SpectraMax readers. With the RiboGreen Fluorescence protocol, SoftMax Pro Software automatically calculated average RFU, standard deviation, and % CV for each set of standard replicates. A standard curve was plotted using the linear curve fit in SoftMax Pro Software (Figure 1A and 1B) for high- and low-range standards run on the SpectraMax iD5, i3x, M5e, and Mini readers. Linearity for each curve was excellent (r20.990).

Sensitivity down to 100 pg/mL was observed using the 96-well microplate format and standard limit of detection calculation of three times standard deviation of the low range blank. This is 10-fold lower than 1 ng/mL stated in the Quant-iT RiboGreen assay product insert and indicates enhanced utility of the assay for applications where sensitivity is critical to success.

High-range (A) and low-range (B) normalized standard curves

***Figure 1.*High-range (A) and low-range (B) normalized standard curves run on 4 different SpectraMax readers. Corected average RFU for each standard was normalized to highest standard RFU (1 μg/mL) for tested plate reader. Curves were plotted using the linear curve fit in SoftMax Pro Software (high-range curves, r20.990; low-range curves, r20.999).

Conclusions

The Quant-iT RiboGreen RNA Assay Kit, when run on a SpectraMax microplate reader with fluorescence detection mode and SoftMax Pro Software, is a rapid, sensitive detection method for RNA. The analysis capabilities of the software provide automated quantitation in an easy-to-read, user-customizable report format.

References

  1. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/dna-rna-quantification
  2. https://www.promega.com/resources/pubhub/choosing-the-right-method-for-nucleic-acid-quantitation/#references-c9a7b496-4311-4517-8968- f79b4eb55b34
  3. https://www.eppendorf.com/product-media/doc/en/59777/Eppendorf_Detection_Application-Note_271_Eppendorf-BioSpectrometer-fluorescence_ Determination-nucleic-acid-concentrations-fluorescent-dyes-Eppendorf-BioSpectrometer-fluorescence.pdf

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