Theory

The quantitation can be performed using any of the following methods:

• UV absorbance (optical density)
• Agarose gel electrophoresis

UV Absorbance for DNA or RNA quantification

The most common and instant technique used to determine both nucleic acid concentration and purity is absorbance. Absorbance measurements can be used to estimate the concentration of DNA or RNA in purified samples.

UV absorbance is measured by small-volume spectrophotometers such as the NanoDrop™ instrument or using a quartz cuvette, which is then placed inside the UV spectrophotometer. The nucleic acid sample is placed either as a drop in a nanodrop or a quartz cuvette carrying nucleic acid samples. Nanodrop, or low-volume quartz cuvette spectrophotometer enables the analysis of sample volumes as low as 1 µL. UV light is passed through the sample at a specified path length, and concentrations of nucleic acids can be directly calculated by measuring absorbance values at 260 nm against a blank using the Beer-Lambert's equation:

UV absorbance (A) = ε x NA concentration x light path length (l)

where:

ε = wavelength-dependent extinction coefficient

c = nucleic acid (NA) concentration

l = light path length (cm)

Some extinction coefficients given for reference*:

• dsDNA (pure): 0.020 (µg/mL)-1 cm-1
• ssDNA (pure): 0.027 (µg/mL)-1 cm-1
• ssRNA (pure): 0.025 (µg/mL)-1 cm-1

*These extinction coefficients do not hold for oligonucleotides or miRNA.

To improve accuracy, the A260 measurement is often corrected for turbidity (measured by absorbance at 320 nm) using the following equation:

Concentration (µg/mL) = (A260 measurement – A320 measurement) x nucleic acid conversion factor** x dilution factor

• Conversion factor for dsDNA: 50 µg/mL
• Conversion factor for ssDNA: 37 µg/mL
• Conversion factor for ssRNA: 40 µg/mL

Total yield can then be obtained by multiplying the nucleic acid concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (mL)

Gel Electrophoresis for DNA AND RNA Quantitation

The most accurate method to quantitate DNA or RNA is to use the combination of absorbance measurements of the sample as mentioned above, and separation of DNA and RNA samples on agarose gel electrophoresis with an appropriate molecular weight ladder.

• A sample of the isolated DNA or RNA is loaded into a well of the agarose gel, which is placed in an electric field. The negatively charged nucleic acid migrates toward the anode, separating DNA and RNA fragments by size and shape.
• Agarose gel electrophoresis visualizes the contaminating bands and sheared impurities within the sample.
• Nucleic acid concentration and yield can be determined by comparing the intensity of sample bands to standards of known amounts.
• Because DNA and RNA absorb light at 260 nm, intensity can be measured using a UV transilluminator. Higher sensitivity can be obtained by labeling nucleic acid samples and standards with a nucleic acid dye such as ethidium bromide or SYBR® Green and measuring intensity at the specified wavelength for that dye.
• If a 2 µL sample of undiluted DNA loaded onto the gel has the same intensity as a 100 ng standard, then the sample concentration is 50 ng/µL (100 ng ÷ 2 µL). Standards used for quantitation should be the same size as the sample nucleic acid being analyzed, and similarly labeled.