Overestimating DNA amounts by UV spectrophotometry? Consider the plastic
Microfuge tubes are one of the most commonly used pieces of labware in molecular biology labs around the globe. While great for holding and storing small samples, the chemicals used in the manufacturing of the plastic could be leading to measurement errors for researchers. Reporting in the current issue of BioTechniques, Kevin Lewis and his colleagues from Texas State University in San Marcos, TX describe their findings that indicate that UV light-absorbing chemicals are capable of leaching from polypropylene microtubes into samples, leading to erroneous measurements of nucleic acid concentration.
The authors surveyed a series of commercially available polypropylene microfuge tube brands under various experimental conditions and found that leaching occurred among all tubes examined. Using mass spectrometry they identified a complex mixture of small molecular weight chemicals that absorbed UV light strongly at 220 and 260 nm, wavelengths normally used to detect and measure protein and DNA concentrations. The leached chemicals resulted in concentration overestimates up to 300%.
"It was quite surprising that leaching of chemical additives which absorb light at the same wavelengths as DNA and proteins had not been described before because it is so ubiquitous among commercially available plastic tubes and biochemists have been using these small plastic tubes for many decades," says Lewis.
Although Lewis and his co-author Gary Beall do not have a solution for the problem, they are encouraged by recent trends in microtube manufacturing. "Some manufacturers have recently begun offering tubes that contain a reduced number of additives," notes Lewis. For studies involving analysis of biological samples by absorbance spectroscopy, Beall and Lewis conclude that at the moment the use of polypropylene tubes with less additives appears to be the best way to reduce measurement errors due to leaching.