DNA and RNA

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.

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

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.

Figure 1. 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.

Table 3. Selection guide for Helixyte™ Green DNA quantifying reagents.

Portelite™ Nucleic Acid Quantitation Kits for Use with the CytoCite™ BG100 and Qubit® Fluorometers*

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 Poretelite™ Quantitation Kits described above, we offer Portelite™ Kits for quantifying endotoxins and proteins.

Advantages of Poretelite™ 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)

Figure 2. 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.

The scope of nucleic acid-based assays continues to expand. There is no consensus on the choice of a method for nucleic acid detection. Key factors governing detection method choice include stability, sensitivity, speed and convenience, and the overall cost for the detection reagents and detection system. The reasons for ultrasensitive analysis in nucleic acid include analysis of genetic material from single cells and from rare cells, such as trophoblasts in maternal circulation, and single-copy gene detection. In addition, better detection methods may provide a route to assays that require fewer amplification cycles or eliminate the multicycle protocols for nucleic acid amplification reactions (e.g., PCR). Although many detection technologies, such as those based on chemiluminescence, may play an important role in the future, at present fluorescence is still the most widely used detection technique in analyzing nucleic acids.

DNA and RNA Quantifying Reagents

Helixyte™ Green dsDNA Quantifying Reagent (Cat# 17597) and StrandBrite™ Green RNA Quantifying Reagent (Cat# 17611) are optimized for double-stranded DNA and single-stranded RNA respectively. They have a high affinity for nucleic acids and an extremely large fluorescence enhancement upon binding nucleic acids, making it possible to directly detect minute amounts of nucleic acids in complex solutions within minutes, usually without interference from other biomolecules.

Advantages of Helixyte™ Green dsDNA and StrandBrite™ Green RNA quantifying reagents:

• High Sensitivity: The Helixyte™ Green and StrandBrite™– based fluorescence assays are up to 10,000-fold more sensitive than UV absorbance measurements.
• Great Selectivity: Unlike measurements of UV absorbance, these assays are not affected by the presence of proteins, free nucleotides or very short oligonucleotides, making quantitation of intact oligonucleotides and nucleic acids much more accurate in complex mixtures, such as serum or whole blood.
• Great Convenience: These assays have a very simple protocol that requires no separation steps, making them ideal for automated, high-throughput measurements.
• Broad Dynamic Range: Quantitation is accurate over four orders of magnitude for the Helixyte™ Green-based assays. The StrandBrite™–based fluorescence assays is accurate over three orders of magnitude.

Product Ordering Information

Table 2. Ordering Info for RNA Quantifying Reagents Products