Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.
The ratio of absorbance at 260 and 280 nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A260/280 ratio when different sources of water were used to perform the spectrophotometric determinations. Adjusting the pH of water used for spectrophotometric analysis from approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA A260/280 ratios from approximately 1.5 to 2.0. Our studies revealed that changes in both the pH and ionic strength of the spectrophotometric solution influenced the A260/280 ratios. In addition, the ability to detect protein contamination was significantly improved when RNA was spectrophotometrically analyzed in an alkaline solution. UV spectral scans showed that the 260-nm RNA absorbance maximum observed in water was shifted by 2 nm to a lower wavelength when determinations were carried out in Na2HPO4 buffer at a pH of 8.5. We found RNA A260/280 ratios to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 in 1-3 mM Na2HPO4 buffer.
Wilfinger
W W
WW
Molecular Research Center, Cincinnati, OH 45212, USA. billw@mrcgene.com
Mackey
K
K
Chomczynski
P
P
D000818:Animals; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D009994:Osmolar Concentration; D010710:Phosphates; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D013053:Spectrophotometry / Q000379:methods*; D014867:Water
D010710:Phosphates; D011506:Proteins; D014867:Water; D012313:RNA; D004247:DNA