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Detection technologies used for DNA and RNA quantification
Nucleic acid quantification (NAQ)—the measurement of DNA or RNA concentration—is a critical step before many downstream experiments. Alongside concentration, assessing sample purity can help to ensure accurate results. Two primary optical methods are used for NAQ: UV-Vis spectrophotometry and fluorescence measurement. Each differs in sensitivity, throughput, sample volume requirements, and the type of information provided. Selecting the right method for your workflow not only improves quantification accuracy but also helps prevent downstream experimental failures, boosting overall efficiency.
Methods of quantification
| Spectrophotometry (UV-Vis) | Fluorescence | |
| How is the optical signal generated? | The photometric measurement of nucleic acids is based on the intrinsic absorptivity properties of nucleic acids (DNA and RNA). When an absorption spectrum is measured, nucleic acids absorb light with a characteristic peak at 260 nm.
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The fluorometric measurement of nucleic acids is based upon the use of fluorogenic dyes that bind selectively to DNA or RNA. These dyes only emit a fluorescent signal when bound to the target.
Fluorescent dyes selectively bind to DNA, RNA or protein. |
| How is the optical signal measured? | The signal is measured by spectrophotometers or spectrometers. The attenuation in the light that reaches the detector after passing through the sample is measured in relation to the incident light and expressed as absorbance values of the sample in the solution. Wavelength separation can take place before or after the light has passed the sample, and the optical light path can be horizontal or vertical.
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The signal is measured by fluorometers . Sample is excited with filtered light (at the excitation wavelength), and the emitted light (at the emission wavelength) is recorded by a detector. Wavelength separation can take place in various ways (for example with filters or with monochromators).
Fluorometer light path. |
| How is the concentration of nucleic acids calculated? | Concentrations of nucleic acids can be directly calculated from their measured absorbance values at 260 nm, using the Beer-Lambert's equation (see below). This concentration calculation is automated in many instruments.
where:
Beer-Lambert equation. |
Concentrations of nucleic acids are measured using the fluorescence signal of the sample and a calibration curve is generated from standard samples of known concentration and fit to appropriate regression models. The limit of detection and linear response of the measurements are specific to each assay.
Typical fluorescence standard curve. |
Featured instruments
We offer a range of instruments to perform nucleic acid quantification using either UV-Vis spectrophotometry or fluorescence measurements.
Benefits and limitations of quantification technologies
Instruments for photometric UV-Vis RNA/DNA quantification
Well-known Thermo Scientific instruments for UV-Vis RNA/DNA quantification include the NanoDrop Ultra Spectrophotometer and Fluorometer for convenient, single-sample microvolume analysis, the NanoDrop Eight Spectrophotometer for 8-sample microvolume analysis, and choice of the Multiskan SkyHigh Microplate Spectrophotometer or the Varioskan ALF and Varioskan LUX Multimode Reader for up to 16 microvolume samples as well as 96- and 384-well plates.
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| Single sample | Eight samples | 6- to 384-well plates, cuvettes, μDrop Plate | 6- to 384-well plates, μDrop and μDrop Duo Plates |
6- to 384-well plates, μDrop and μDrop Duo Plates |
 NanoDrop Ultra Microvolume UV-Vis Spectrophotometers |
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 Varioskan LUX Multimode Microplate Reader |
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| Lower detection limit (dsDNA) | Pedestal: 1.0 ng/µL Cuvette: 0.2 ng/µL | 2.0 ng/µL | Depends on sample format and volume used | Depends on sample format and volume used | Depends on sample format and volume used |
| Upper detection limit (dsDNA) | Pedestal: 27,500 ng/µL Cuvette: 75 ng/µL | 10,000 ng/µL | Depends on sample format and volume used | Depends on sample format and volume used | Depends on sample format and volume used |
| Sample formats | Drop (1 µL) cuvettes | Drop (1 µL) | 6–384 well plates µDrop Plate (2–10 µL) cuvettes (not all models) | 6–384 well plates µDrop and µDrop Duo Plates (2–10 µL) | 6–384 well plates µDrop and µDrop Duo Plates (2–10 µL) |
| Throughput | One sample at a time | Up to 8 | One sample at a time with cuvettes Up to 16 with µDrop plate Up to 96 with 96-well plates Up to 384 with 384 well plate |
Up to 16 with µDrop plate Up to 96 with 96-well plates Up to 384 with 384 well plate |
One sample at a time with cuvettes Up to 16 with µDrop plate Up to 96 with 96-well plates Up to 384 with 384 well plate |
| Sample volume | 1 µL | 1 µL | As little as 2 µL depending on assay format | As little as 2 µL depending on assay format | As little as 2 µL depending on assay format |
| Processing time | ≤7 seconds | <20 seconds for 8 samples | ~1 minute for 16 microvolume samples in the µDrop Plate | ~1.1 minute for 16 microvolume samples in the µDrop Plate | ~1.3 minutes for 16 microvolume samples in the µDrop Plate |
| Spectral data | Yes | Yes | Yes | Yes | Yes |
| Sample purity information | Yes, it provides full spectral data (A260/A280 and A260/A230 ratios) as well as Acclaro Contaminant Analysis | Yes, it provides full spectral data (A260/A280 and A260/A230 ratios) as well as Acclaro Contaminant Analysis | Yes, it provides full spectral data (A260/A280 and A260/A230 ratios) | Yes, it provides full spectral data (A260/A280 and A260/A230 ratios) | Yes, it provides full spectral data (A260/A280 and A260/A230 ratios) |
| Contaminants detection | Yes, Acclaro Sample Intelligence technology detects protein, phenol and guanidine salts and it gives true concentration of the nucleic acids | Yes, Acclaro Sample Intelligence technology detects protein, phenol and guanidine salts and it gives true concentration of the nucleic acids | No, some information is available, based on the purity ratios | No, some information is available, based on the purity ratios | No, some information is available, based on the purity ratios |
| Distinguish between DNA and RNA | Yes, distinguishes between mammalian DNA/RNA, bacterial DNA/RNA, and plant DNA/RNA | Yes, distinguishes between mammalian DNA and mammalian RNA | No | No | No |
| Oligonucleotide quantification | Yes, with embedded applications and custom methods | Yes, with embedded applications and custom methods | Yes | Yes | Yes |
| Pre-configured protocols | Yes, there are user interface touch screen and custom protocols | Yes, there are user interface touch screen and custom protocols | Yes, there are pre-configured protocols in the interface touch screen and SkanIt software Cloud Library, free with SkanIt Software | Yes, there are pre-configured protocols in the SkanIt software Cloud Library, free with SkanIt Software | Yes, there are pre-configured protocols in the SkanIt software Cloud Library, free with SkanIt Software |
| Thermo Fisher Connect | Yes | No | Yes | No | No |
| 21 CFR Part 11 | Yes, with optional software | Yes, with optional software | Yes, with SkanIt Software Drug Discovery Edition | Yes, with SkanIt Software Drug Discovery Edition | Yes, with SkanIt Software Drug Discovery Edition |
| Robotic automation compatibility | No | No | Yes | Yes | Yes |
| User interface | Via a touch screen user interface or via the NanoDrop Ultra PC control Software | With PC software (Nanodrop Eight Operating Software) | Via a touch screen user interface or the PC Software (SkanIt Software) | Via PC software (SkanIt Software) | Via PC software (SkanIt Software) |
Instruments for fluorescence RNA/DNA quantification
High-performing instruments for performing fluorescence DNA or RNA quantification include Thermo Scientific NanoDrop Ultra FL/FL-C and Invitrogen Qubit 4 Fluorometers for single-sample measurements, Invitrogen Qubit Flex Fluorometer for medium-throughput measurements and the Thermo Scientific Varioskan ALF and Varioskan LUX Multimode Microplate Reader for 96- to 1536-well measurements.
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| Single sample | Single sample | Eight samples | 6- to 384-well plates, μDrop and μDrop Duo Plates |
6- to 1536-well plates, μDrop and μDrop Duo Plates |
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| Lower detection limit (dsDNA) | 0.05 ng/µL*** | 0.005 ng/µL* | 0.005 ng/µL* | Depends on the Quant-iT assay kit, standard concentrations and curve fittings used** 0.005 ng/µL | Depends on the Quant-iT assay kit, standard concentrations and curve fittings used** 0.005 ng/µL |
| Upper detection limit (dsDNA) | 1,500 ng/µL**** | 4000 ng/µL* | 4000 ng/µL* | Depends on the Quant-iT assay kit and assay conditions | Depends on the Quant-iT assay kit and assay conditions |
| Sample formats | Sample added directly to instrument | Qubit assay tubes | Qubit Flex tube strips | 6- to 384-well plates, μDrop and μDrop Duo Plates |
6- to 1536-well plates, μDrop and μDrop Duo Plates |
| Throughput | 1 | 1 | Up to 8 | Dependent on assay format | Dependent on assay format |
| Sample volume | 1–10 µL | 1–20 µL | 1–20 µL | Dependent on assay format | Dependent on assay format |
| Processing time | ≤10 seconds | <3 seconds | ~3 seconds for 8 samples | Dependent on assay format | Dependent on assay format |
| Sample purity and contaminants detection | No, only through absorbance applications | No | No | No | No |
| Distinguish between DNA and RNA | Yes, using fluorescent dyes that are specific for DNA or RNA | Yes, using fluorescent dyes that are specific for DNA or RNA | Yes, using fluorescent dyes that are specific for DNA or RNA | Yes, using fluorescent dyes that are specific for DNA or RNA | Yes, using fluorescent dyes that are specific for DNA or RNA |
| RNA quality and integrity score | No | Yes, when used with Qubit RNA IQ Assay Kit | Yes, when used with Qubit RNA IQ Assay Kit | No | No |
| Oligonucleotide quantification | No | Yes, with ssDNA assay kit | Yes, with ssDNA assay kit | Yes, with Quant-iT OliGreen assay kit | Yes, with Quant-iT OliGreen assay kit |
| Pre-configured protocols | Yes, there are user interface touch screen and custom protocols | Yes, there are user interface touch screen and custom protocols | Yes, there are pre-configured protocols in the interface touch screen and SkanIt software Cloud Library, free with SkanIt Software | Yes, there are pre-configured protocols in the SkanIt Software Cloud Library, free with SkanIt Software | Yes, there are pre-configured protocols in the SkanIt Software Cloud Library, free with SkanIt Software |
| Thermo Fisher Connect | Yes | Yes | Yes | No | No |
| 21 CFR Part 11 | Yes, with optional software | No | Yes, with optional software | Yes, with SkanIt Software Drug Discovery Edition | Yes, with SkanIt Software Drug Discovery Edition |
| Automation | No | No | No | Yes | Yes |
* Reported here are the lower and upper quantitation ranges measured with Qubit dsDNA High sensitivity (HS) kit (Cat. No. Q32851) Qubit dsDNA Broad Range (BR) kit (Cat. No. Q32850) respectively and using variable sample volume (1–20 µL).
** Limit of Detection (LOD) using the Quant-iT dsDNA Assay HS kit (Cat. No. Q33120) as instructed. LOD estimated according to IUPAC, based on the precision of the blanks (3*SD). Results will vary depending on the curve fittings, standard concentrations and used kits.
*** Reported here is the lower quantitation range measured with the NanoDrop Ultra dsDNA BR Fluorescence Assay and using sample volume (10 µL).
**** Reported here is the upper quantitation range measured with the NanoDrop Ultra dsDNA BR Fluorescence Assay and using sample volume (1 µL).
Featured instruments, kits, and reagents
UV-Vis spectrophotometers
Fluorometers
Combined UV-Vis Spectrophotometer and Fluorometer
FAQ
A: UV and fluorescence technologies work differently to quantify DNA. UV quantification relies on the intrinsic absorptivity of DNA and RNA molecules, while fluorescence quantification uses dyes that specifically bind to your molecule of choice. With UV technology, quantification isn't as sensitive, but it has a broader dynamic range and also gives data about sample purity--plus it is faster because there is no reagent prep. With fluorescence technology you get higher sensitivity and molecule-specific data, but it has lower dynamic range and reagent prep is required. The technology you need will depend on what features are important for your lab and experimentation.
A: All of the DNA instruments offered by Thermo Fisher Scientific are of excellent quality. To find the right instrument to quantify DNA or RNA in your lab, explore the detailed comparison of UV-Vis spectrophotometers and fluorometers. Things like sensitivity, throughput and budget may be initial considerations when selecting a DNA quantification instrument. You may also consider whether you need target specificity, sample purity information, RNA quality information, or broad dynamic range.
Resources
Related pages
For Research Use Only. Not for use in diagnostic procedures.