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Plastics hamper DNA assays

April 23, 2010 By Alla Katsnelson This article courtesy of Nature News.

Chemicals leaching from lab plastic throw off results.

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Biologists using standard plastic test tubes to gauge the concentration of DNA and proteins in their samples may be getting wildly incorrect readings because chemicals are leaching out of the containers.

An established way of assessing the concentrations and some key properties of DNA and proteins is to measure the levels of ultraviolet light the molecules absorb at wavelengths of between 220 and 260 nanometres. But methods used routinely in molecular biology cause the plastic tubes to release compounds that absorb light in the same range, increasing the reading by as much as 300%, say researchers from Texas State University in San Marcos who have studied the problem1.

"The leaching of chemicals out of polypropylene tubes consistently resulted in an overestimation of measurements," says Kevin Lewis, a molecular geneticist and an author on the study.

Graduate student Michael Robson stumbled on the effect while studying how efficiently DNA binds to clay. To his surprise, water centrifuged for 30 minutes or longer — during which time it warmed up slightly — began to absorb light at wavelengths characteristic of DNA. Suspecting that their microcentrifuge tubes might be the culprits, Robson and his colleagues took a closer look at ten types of tube from nine manufacturers, using mass spectrometry to determine the levels of leached chemicals. Leaching increased both after the tubes were heated above 37 °C and when inorganic solvents were used — procedures used in enzyme-catalysed reactions, protein extractions and the polymerase chain reaction.

There is "no doubt", Lewis says, that some results in the literature are incorrect, at least quantitatively — although the relative differences between control and experimental samples should still hold. He adds that double-checking results with other techniques, such as gel electrophoresis or fluorometry, in which leaching chemicals do not affect readings, would catch the problem.

Widespread problem

Manufacturers contacted by Nature were not able to respond before publication of this story. But the Texas team isn't alone in reporting concerns over lab plastics. In November 2008, researchers in Canada found that chemicals in pipette tips and plastic tubes interfered with the activity of an enzyme they were studying2 (see 'Why plastic isn't always fantastic'). And more recently, researchers in Germany reported that standard-issue Petri dishes — made of polystyrene, another type of plastic — were skewing the results of cell-culture assays3.

"Despite increasing exposure of this problem over the past two or three years, pipette tips and microfuge tubes purchased from the majority of sources — and particularly the more inexpensive brands — still contain additives that leach into buffers and solvents and interfere with measurements," pharmacologist Andrew Holt of the University of Alberta in Edmonton, Canada, told Nature.

The problem has also found its way into the clinic. Last year, Holt and his colleagues at Hadassah-Hebrew University Medical Center in Jerusalem reported a spike in diagnoses of a rare metabolic disorder after the lab changed the supplier of its pipette tips4.

Lewis's lab, which reports its results in the April issue of BioTechniques1, now uses only tubes advertised as low in additives. "There may be some drawbacks" to such tubes, he notes, because the additives are meant to protect the plastic. "They may break down or become brittle faster with time."

References

  1. Lewis, L. K. et al. BioTechniques 48, 297-302 (2010).
  2. McDonald, G. R. et al. Science 322, 917 (2008).
  3. Sommer, A. P. et al. J. Bionic Eng. 7, 1-5 (2010).
  4. Belaiche, C. et al. Clin. Chem. 55, 1883-1884 (2009).

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