Accurate nucleic acid quantitation is an essential step in DNA and RNA sample preparation and regularly contributes to the success (or failure) of various downstream molecular experiments. Methods commonly used to quantify DNA and RNA include absorbance (UV-Vis) measurements, fluorescence measurements, and real-time polymerase chain reaction (qPCR)
. While each method presents its advantages, choosing the right technology can save significant time and money and depends mainly on the experimental need and the intended downstream application.
For efficient and effective nucleic acid quantitation, AAT Bioquest offers various products, including hand-held fluorometers, Helixyte™ and Portelite™ kits for the fluorescent determination of DNA or RNA concentration, and DNA binding dyes and probes
for qPCR based quantitation.
Absorbance versus Fluorescence Nucleic Acid Quantitation
Traditionally, DNA and RNA concentrations are quantified by measurement of light absorption via UV-VIS spectrophotometry. Absorbance is measured at 260 nm, where both DNA and RNA absorb light most strongly, and the total nucleic acid concentration is determined using a specific conversion factor. Additionally, measurements taken at 230, 280, 320, or 340 nm can be used to detect non-nucleic acid contaminants, such as proteins, organic materials, and other interfering compounds. While absorbance methods are advantageous for their simplicity, ability to detect contaminants, and provide information on sample purity, they cannot, however, distinguish between DNA or RNA because both absorb light at 260 nm.
Fluorescence-based quantitation methods using fluorescent dyes that selectively bind to double-stranded DNA, single-stranded DNA, or RNA, such as Helixyte™ Green dsDNA
, Helixyte™ Green ssDNA
, or StrandBrite™ Green RNA dyes
, allow for more sensitive and specific determination of nucleic acids over the conventional UV-Vis spectroscopy method. The fluorescent signal, which is measured by fluorometers, is proportional to the amount of nucleic acid present in the sample, and because fluorescent dyes bind in a highly specific manner, measurements are accurate despite possible contamination. A key difference between these two methods is that quantitation via fluorescence is an indirect method, and therefore requires the generation of a calibration curve from standard samples of known concentration before quantitation.
Table 1. Comparison of absorbance and fluorescence-based nucleic acid quantification methods
- Quick and easy-to-use
- Non-hazardous - does not require any reagents
- Provides information on sample purity
- Limited low-end sensitivity
- Inaccuracies introduced by contaminants
- Cannot distinguish between ssDNA, dsDNA, or RNA
- No reagents needed for quantification
- Quick and easy-to-use
- Highly sensitive and accurate
- Ideal for the analysis of low-level samples
- Contaminants pose minimal effect on results
- Preferential detection of nucleic acid species
- Requires calibration with standard samples
Quantifying Nucleic Acids in Solution
AAT Bioquest offers an extensive portfolio of highly sensitive nucleic acid binding dyes and fluorescence-based assays to quantify nucleic acids in solution. The Helixyte™ Green dsDNA reagent, Helixyte™ Green ssDNA reagent, and StrandBrite™ Green RNA reagent - optimized for double-stranded DNA, single-stranded DNA (e.g., oligonucleotides), and RNA, respectively - bind selectively and with high affinity to their nucleic acid target of interest. These dyes exhibit a considerable fluorescence enhancement upon binding nucleic acids, enabling the rapid detection of low concentrations of nucleic acids in complex solutions without interference from other biomolecules. The Portelite™ Quantitation Kits
are designed to deliver high sensitivity and selective quantitation of ssDNA, DNA, RNA, or total nucleic acid concentrations using the CytoCite™ BG100 and Qubit™ fluorometers. Each Portelite™ assay has been specially formulated with a target-selective nucleic acid dye, ready-to-use buffers, DNA standards, and an optimized protocol, allowing for accurate and straightforward quantitation using a simple mix-and-read format.
Helixyte™ Green DNA Quantitation Kits and Stand-Alone Reagents
Comparison of dsDNA dose-response using the Helixyte Green™ Fluorimetric dsDNA Quantitation Kit *High Sensitivity* (blue) with Quant-iT™ PicoGreen® dsDNA Assay Kit (red ). dsDNA standards were incubated in cuvettes and measured using a Varian Cary Eclipse fluorescence spectrophotometer.
The Helixyte™ Green series of assay kits and stand-alone reagents are designed for simple, accurate and sensitive determination of ssDNA, dsDNA, or total nucleic acid concentration in solution using a standard fluorescence microplate reader. Each Helixyte™ assay kit is specially formulated with a target-selective DNA dye, a ready-to-use buffer, pre-diluted DNA standards, and an optimized protocol, allowing accurate quantitation across a wide range of concentrations with minimal interference from common contaminants. Unlike absorbance measurements, these assays are several orders of magnitude more sensitive over a wide linear detection range and are ideally suited for high-throughput quantitation using 96- or 384-well plates. The Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *High Sensitivity* can be used to detect as little as 25 pg/mL of dsDNA in complex solutions, making it ideal for quantitating genomic DNA, viral DNA, miniprep DNA, PCR amplification products, and other applications requiring low volumes of DNA.
Table 2. Helixyte™ Green Fluorimetric DNA Quantitation Kits for use with fluorescence microplate readers.
|Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Broad Dynamic Range*||503||527||dsDNA||2-1000 ng||200 tests||17645|
|Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Broad Dynamic Range*||503||527||dsDNA||2-1000 ng||1000 tests||17646|
|Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Microplate Readers*||502||522||dsDNA||0.2-100 ng||200 tests||17650|
|Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *High Sensitivity*||502||522||dsDNA||0.2-100 ng||200 tests||17651|
|Helixyte™ Green Fluorimetric ssDNA Quantitation Kit *Optimized for Microplate Readers*||498||519||ssDNA||1-200 ng||200 tests||17623|
|Helixyte™ Green Fluorimetric Total Nucleic Acid Quantitation Kit *Optimized for Microplate Readers*||509||529||dsDNA, ssDNA, RNA, oligos||as little as 0.3 ng||200 tests||17630|
Table 3. Selection guide for Helixyte™ Green DNA quantifying reagents.
|Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*||490/525||dsDNA||0.2-100 ng||1 mL||17597|
|Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*||490/525||dsDNA||0.2-100 ng||10 mL||17598|
|Helixyte™ Green ssDNA Quantifying Reagent||490/525||ssDNA||1-200 ng||1000 tests||17620|
|Helixyte™ Green ssDNA Quantifying Reagent||490/525||ssDNA||1-200 ng||10000 tests||17621|
|Helixyte™ 14 [Equivalent to SYBR 14]||489/520||DNA|| ||1 mg||17564|
|Helixyte™ 14 [Equivalent to SYBR 14] *5 mM*||489/520||DNA|| ||0.5 mL||17563|
Portelite™ Nucleic Acid Quantitation Kits for Use with the CytoCite™ BG100 and Qubit® Fluorometers*
DNA standard curve generated using Portelite™ Fluorimetric DNA High Sensitivity Quantitation Kit (Cat No. 17661). Fluorescence intensity was quantified using the FITC channel, and the regression model was calculated using log-log best-fit. The detection limit was 10 pg/µL.
Portelite™ Fluorimetric Quantitation Kits, optimized for the CytoCite™ BG100 Fluorometer, are designed to selectively quantify total nucleic acid, DNA (ssDNA, dsDNA), and RNA, providing more sensitive analysis than comparable UV absorbance-based instrumentation. The aforementioned kits are suitable for a variety of common biological applications, including cloning, sequencing, transfection, qPCR, and gel electrophoresis
. All assays are performed using the same general protocol, providing a simple mix-and-read assay format for minimal hands-on time. Each Portelite™ kit contains a concentrated assay reagent, dilution buffer, and pre-diluted standards. The fluorescent probes in each kit have similar spectral properties, being blue light-excitable (480 nm), with emissions between 510 nm and 580 nm. While developed specifically for use with the CytoCite™ BG100 fluorometer, Portelite™ Fluorometric kits may also be used on other commercially available compact fluorometric instruments, such as the Qubit®
fluorometer. In addition to the Portelite™ Quantitation Kits described above, we offer Portelite™ Kits for quantifying endotoxins
Advantages of Portelite™ Fluorimetric Quantitation Kits
- Enhanced selectivity - assays utilize target-specific dyes that bind selectively to ssDNA, dsDNA, total nucleic acid, or RNA
- High sensitivity - requires as little as 1 µL of sample for quantitation
- Wide dynamic range - fluorescence-based assays with 4 to 5 orders of magnitude linear dynamic range (10 pg/µL to 1 µg/µL DNA)
Table 4. Portelite™ Fluorimetric Nucleic Acid Quantitation Kits for use with the CytoCite™ and Qubit® fluorometers.
|Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit||CytoCite™ BG100 or Qubit® Fluorometer||480/520||dsDNA||0.2-100 ng||100 tests||17660|
|Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit||CytoCite™ BG100 or Qubit® Fluorometer||480/520||dsDNA||0.2-100 ng||500 tests||17661|
|Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range||CytoCite™ BG100 or Qubit® Fluorometer||480/530||dsDNA||2-1000 ng||100 tests||17665|
|Portelite™ Fluorimetric ssDNA Quantitation Kit||CytoCite™ BG100 or Qubit® Fluorometer||480/530||ssDNA||1-200 ng||200 tests||17625|
|Portelite™ Fluorimetric Total Nucleic Acid Quantitation Kit||CytoCite™ BG100 or Qubit® Fluorometer||480/530||dsDNA, ssDNA, RNA, oligos||as little as 0.3 ng||200 tests||17631|
|Portelite™ Fluorimetric RNA Quantitation Kit||CytoCite™ BG100 or Qubit® Fluorometer||480/530||RNA||5-100 ng||100 tests||17658|
|Portelite™ Fluorimetric RNA Quantitation Kit||CytoCite™ BG100 or Qubit® Fluorometer||480/530||RNA||5-100 ng||500 tests||17659|