Nucleic acid quantification

Nucleic acid quantification

High accuracy, low volume nucleic acid quantification

Dye-based DNA and RNA quantification is common, but gets bogged down with lots of steps and expensive reagents. If you have a high concentration sample, it may be outside the linear range of a fluorescent dye assay – then you’re faced with the impact serial dilutions have on accuracy. UV/Vis spectroscopy on Big Lunatic and Stunner equips you with high-throughput total nucleic acid quantification at low volumes, without any additional reagents or serial dilutions. DNA and RNA quants on Big Lunatic and Stunner deliver spot-on accuracy within 2% and precision within 1% over a dynamic range that covers low and high concentrations of double-stranded or single-stranded DNA and RNA.

Figure 1: Measured absorbance for a dilution series of DNA and RNA.

Figure 1: Measured absorbance for a dilution series of DNA and RNA.

Nucleic acid absorbance spectra have a peak at 260 nm in the UV range (Figure 1). This A260 value is directly proportional to the nucleic acid concentration, c, the pathlength through the sample, l, and an extinction coefficient, ε. This relationship is described by Beer’s law, also called the Beer-Lambert law:

 

For nucleic acids, Beer’s law is often simplified and rearranged into:

 

Here, F is a concentration factor which varies depending on the nucleic acid. You need 50 ng/µL dsDNA measured with a 1 cm pathlength to get an A260 value of 1 OD. Therefore, the concentration factor F for dsDNA is 50, while for ssDNA it is 33 and for RNA it is 40.

If you’re working with oligonucleotides, Lunatic and Stunner have built-in calculators to determine sequence-specific concentration factors for RNA or DNA oligo quantification. Just copy in your sequence and out come mass and molar concentrations.

Lunatic and Stunner combine Beer’s law with exceptionally accurate absorbance measurements in fixed pathlength microcuvettes to get crazy good accuracy and precision of nucleic acid quantification (Figure 2).

Figure 2: Known vs. measured concentration averages for a DNA (left) and RNA (right) dilution series.

Figure 2: Known vs. measured concentration averages for a DNA (left) and RNA (right) dilution series.

A260 is great for quantifying purified nucleic acids, but what if you have a sample of unknown quality? Lunatic and Stunner have the tools to quality check your samples before they move to the next step in the workflow. A260/A280 and A260/A230 ratios can be used to tell the purity of a sample of DNA or RNA.

DNA typically has A260/A280 of between 1.7–1.8, while good quality RNA will have an A260/A280 ratio ≥2.0. Lower A260/A280 values may indicate contamination by protein, phenol, or other aromatic compounds.

The A260/A230 ratio of pure DNA should be ≥1.8. Lower values can indicate the presence of impurities including EDTA, guanidine, carbohydrates, or proteins.

Lunatic and Stunner utilize the full potential of UV/Vis spectroscopy with their Unmix applications. These applications analyze complex UV/Vis spectra to look for RNA, DNA, or protein, along with impurities commonly found in these samples. As a result, a spectrum can be deconvoluted to show the contribution from each component within a solution (Figure 3). This allows for specific quantification of a biomolecule of interest, like DNA, in the presence of certain co-absorbing impurities. It can also make it easier to troubleshoot complex samples and evaluate sample purity more clearly than you would get with absorbance ratios alone.

Figure 3. Unmix deconvoluted spectra of DNA and other impurities, including 5 mM EDTA (left). Unmix deconvoluted spectra of DNA and other impurities, including protein (right).

Figure 3. Unmix deconvoluted spectra of DNA and other impurities, including 5 mM EDTA (left). Unmix deconvoluted spectra of DNA and other impurities, including protein (right).

UV/Vis spectroscopy is one of the oldest and most well-established techniques to quantify DNA or RNA and determine nucleic acid quality. Unchained Labs pushes UV/Vis to the next level with innovative optics, optimized consumables and robust analytical techniques.

Big Lunatic and Stunner deliver highly accurate nucleic acid quantification with A260 values from 0.03–275 OD quickly and without dilution. That means you can measure dsDNA concentrations between 1.5–13,750 ng/µL for up to 96 samples in 10 minutes. With Unmix applications or A260/A280 and A260/A230 ratios you can determine sample quality and unravel even the dirtiest samples. If you need more information on UV/Vis spectroscopy for nucleic acids, check out this tech note all about DNA quantification and quality.

Unchained Labs UV/Vis instruments deliver:

  • Accurate and precise full-spectrum UV/Vis absorbance results from 230–750 nm
  • Results from tiny 2 µL sample volumes
  • Wide dynamic range from 0.03 to 275 OD
  • dsDNA quantification from 1.5–13,750 ng/µL
  • 96 UV/Vis reads in about 10 minutes
  • Purpose-built applications for nucleic acid quantification of any sample
  • Simple A260/A280 and A260/A230 quality control metrics
  • Microfluidic circuits to protect samples from evaporation and cross-contamination
  • Fixed pathlength microcuvettes for maximum sensitivity and reproducibility
  • Low maintenance and no clean-up
  • Performance Verification for USP and Ph. Eur. Compliance

Big Lunatic

Big Lunatic makes batch quantification of RNA or DNA a no-brainer. All you need is 2 µL and 10 minutes to measure up to 96 samples. Run them straight-up, even at high concentrations, without ever diluting. Measurements are spot-on with precision within 1% and accuracy within 2% and are fully compliant with USP and Ph. Eur. guidelines. The SBS plate-based format means integrating with lab automation and liquid handlers is a snap. Big Lunatic gets biologics UV/Vis quantification on the money every time. Just drop, load, and read.

Stunner

Want to check if your sample is now a blob of aggregates? Stunner combines Big Lunatic’s plate-based, high-throughput UV/Vis accuracy and precision with dynamic light scattering for a one-of-a-kind combination. Pairing up these techniques checks concentration, hydrodynamic size, polydispersity, and detection of aggregates off your to-do list in one shot. Prove that you can trust the data and the sample you hand off to the next scientist.

Stunner

Little Lunatic

Little Lunatic is there for labs that have fewer samples and lower requirements. It uses a chip-based version of the Big Lunatic plate to get quick concentration measurements of up to 16 samples in 5 minutes. Researchers that just need a quick check of concentration can get what they need from this little guy and its glove-friendly touch screen.

Little Lunatic

More info

Have a question or can’t wait to find out more? Here’s the best way to reach us: