How to Quantify DNA and RNA

Methods for Quantifying DNA and RNA

The most common methods for quantifying DNA or RNA use UV absorbance or fluorescence. Here we offer a brief overview of how these methods work, how they differ from each other, and how to perform them.

How to quantify DNA or RNA using UV absorbance

UV absorbance is one of the most—if not the most—common ways to quantify DNA and RNA because it is quick, simple, and can provide some insight into the sample quality. However, the method cannot distinguish between DNA and RNA, it is not sensitive enough to detect low concentrations, and accuracy is limited when contaminants are present.

How it works:

This method involves measuring the absorbance of light at a particular wavelength through a liquid (e.g., water or buffer) to determine the concentration of DNA and RNA in the liquid. While nucleic acids absorb maximally at 260 nm, several contaminants also absorb strongly at wavelengths near 260 nm. Further, the presence of organic solvents and proteins can inflate the reading at 260 nm, producing an inaccurate DNA or RNA concentration.

Wavelengths to remember:

• Nucleic acids maximally absorb at 260 nm
• Proteins maximally absorb at 280 nm
• Organic compounds and chaotropic salts maximally absorb at 230 nm

To measure the concentration of DNA or RNA in your sample, you’ll need a spectrophotometer using UV-transparent cuvettes, or you can use a microvolume spectrophotometer, such as a NanoDrop.

Using a spectrophotometer is an easy three-step process:

1. Dilute your DNA or RNA in nuclease-free water or buffer (e.g., TE buffer)
2. Measure your blank at 260 nm: water or buffer without DNA/RNA to measure the background absorbance
3. Measure the absorbance of the diluted DNA or RNA sample at 260 nm (A260)

The concentration of DNA or RNA is based on Beer-Lambert's equation (conveniently, many instruments automatically calculate this): A=εCL

• C = nucleic acid concentration in molar (M)
• A = UV absorbance in absorbance units (AU)
• ε = wavelength-dependent molar absorptivity coefficient (or extinction coefficient) in M^-1cm^-1
• L = light path in cm (cm)

If your instrument doesn’t automatically calculate the concentration, you can use the equations and key values below to get a fairly accurate estimate:

• DNA Concentration (µg/mL) = A260 reading x dilution factor x 50 µg/mL
• RNA Concentration (µg/mL) = A260 reading x dilution factor x 40 µg/mL

Summary of key values

How to quantify DNA or RNA using Fluorescence

Fluorescence-based methods are sensitive and suitable for detecting low concentrations, making them useful for quantifying DNA or RNA for sequencing. Another distinction from spectrophotometric methods is that you can distinguish between DNA and RNA, and contamination only minimally affects the results.

How it works:

This method involves fluorescent dyes that selectively bind to DNA or RNA, and when the dye is bound to the target it emits a signal that gets detected by a fluorometer.

To calculate the concentrations of nucleic acids in your sample, you’ll first need to generate a calibration curve using standard samples of known concentration. While this takes some time upfront, it is quick and easy to use when you need to measure your sample concentrations in the future.

Once you have a reliable standard curve, you can compare the fluorescence of your sample against the curve to quantify your DNA or RNA—many fluorometers will calculate your sample concentration for you.

Alternative methods for quantifying DNA or RNA

There are several other methods for determining the concentration of DNA or RNA, such as gel electrophoresis and real-time PCR (qPCR).

Compared to UV absorbance and fluorescence-based methods, these alternative methods will require substantially more time to set-up, costly reagents, and advanced instrumentation. However, they offer several advantages. Electrophoresis enables you to evaluate the size and quality of your DNA or RNA and real-time PCR is incredibly sensitive and can accurately quantitate subsets of specific nucleic acids based on sequence.

Summary

Measuring the concentration of DNA and RNA in your sample is a critical step in any molecular biology lab that can help optimize sample preparation as well as ensure consistency and reproducibility of downstream applications. There are several methods you can choose from—each having advantages and disadvantages—and oftentimes it is beneficial to use multiple throughout your workflow.