Download PDF

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

• Quant-iT RiboGreen RNA Assay Kit (Invitrogen cat. #R11490)
• 96-well solid black microplate (Greiner Bio-One cat. #655076)
• Light Safe black microtubes (Argos cat. #T7100BK)
• Microplate readers
  • SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices P/N iD5)
  • SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices P/N i3x)
  • SpectraMax® M5e Multi-Mode Microplate Reader (Molecular Devices P/N M5e)
  • SpectraMax® Mini Multi-Mode Microplate Reader (Molecular Devices P/N SMAX MINI AF) with fluorescence filter cube FL-535 (Molecular Devices P/N 5089097)

Methods

Instrument and protocol setup

• Turn on the microplate reader.
• Launch SoftMax® Pro Software and open the RiboGreen Fluorescence protocol from the Nucleic Acids folder in the Protocol Library dropdown menu of the Protocol Manager. Depending on which SpectraMax reader is used, select optimized settings for the assay (see Table 1).
• Select wells to read and assay plate type by clicking on “Acquisition Settings” and locating the options on the left side of the screen.
• Click the “Template Editor” button to assign wells of the microplate to pre-set template groups. There are preconfigured template groups in the RiboGreen Fluorescence protocol including Standards, Unknowns, and Unknowns_Diln (use for samples that are diluted). You may add unique Standard groups for High-Range Standards and Low-Range Standards by selecting Add.

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.

• Prepare 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) by diluting the concentrated buffer from the kit 20-fold with distilled DEPC-treated water.
• Prepare aqueous working solutions of Quant-iT RiboGreen reagent by making a 200-fold dilution (for high-range assay: 20 ng/mL to 1 μg/mL RNA) and 2000-fold dilution (for low-range assay: 1 ng/mL to 50 ng/mL RNA) of the concentrated DMSO solution in 1 X TE buffer (prepared above). Prepare solutions in 15-mL conical tubes, rather than glass, to avoid reagent adsorption to glass surfaces. Protect the solution from light by covering with foil and protecting from light until ready to use.
• High-range RNA standard curve: Prepare a 2 μg/mL stock solution of RNA in TE. The lambda RNA standard provided with the kit is diluted 50-fold in TE to make the 2 μg/mL solution. For this application note, a series of standards ranging from 20 ng/mL to 1 μg/mL were prepared per Table 2 in Light-Safe black microtubes.
• Low-range RNA standard curve: Prepare a 100 ng/mL stock solution of RNA in TE. The 2 μg/mL RNA stock solution is diluted 20-fold in TE to make the 100 ng/mL solution. For this application note, a series of standards ranging from 0.0008 ng/mL to 25 ng/mL were prepared per Table 3a and 3b. Table 3a depicts the serial dilution to obtain low-range working dilutions prepared in RNAse-free microfuge tubes. Table 3b depicts the final RNA concentration after 500 μL of 2000-fold diluted Quant-iT RiboGreen reagent was added to low-range dilution in Light-Safe black microtubes. This stepwise approach for preparing low-range standards was executed to assess the lower limit of detection on SpectraMax readers compared to the indicated kit sensitivity of 1 ng/mL.
• Pipet standards in working solution into a solid black 96-well microplate at 200 μL per well, in triplicate; including triplicate blank wells containing TE only (no RNA).
• Incubate for 2 minutes at room temperature, protected from light.

Read the microplate

• Place the microplate in the drawer. For SpectraMax M-Series readers, place the microplate on top of the purple plate adapter in the plate carriage.
• Click the Read button in SoftMax Pro Software.

Analyze the data

• After the microplate was read, group blanks are automatically subtracted from the data. the corrected relative fluorescence units (RFUs) are displayed in the Plate section. The data are analyzed in the Group Tables created when the template was set up.
• Corrected RFU for each standard read on each plate reader are normalized to that of the highest standard (1 μg/mL) and plotted in the High-Range Standard Curve and Low-Range Standard Curve sections of the experiment. A linear curve fit is selected from the dropdown menu in the graph section.

***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.

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

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

***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.

***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

Download PDF