Significance of 260/230 ratio

06/23/04

The Significance of the 260/230 Ratio in Determining

Nucleic Acid Purity
Nucleic acids only absorb light that has a wavelength of 260 nm. Organic
contaminants like phenol and other aromatic compounds, TRIzol, and some
reagents used in RNA extraction absorb light of a 230 nm wavelength. Samples
with a low 260/230 (below about 1.8) have a significant presence of these
organic contaminants that may interfere with other downstream processes like
RT-PCR or the IVT in Affymetrix experiments, lowering their efficiency. In
order to foster the success of the microarray and gene expression experiments
performed at the Microarray Core Facility, we strongly recommend limiting
microarray experiments to those samples with a 260/230 ratio greater than 1.8.
Running samples with 260/230 ratios lower than 1.8 could have reduced yield
and substantially less optimal results. MCF users who wish to run samples that
have organic contamination indicated by a low 260/230 ratio must assume
responsibility for the performance of their samples. The MCF requires that in
order to run samples with low 260/230 ratios, the user must sign off on the
samples showing that he/she is aware of the risk associated with performing
microarray experiments with samples that do not the meet sample purity
standards delineated by the MCF and supported by Affymetrix. Below are links
to various Internet sources that speak to the significance of the 260/230 ratio in
discerning organic contamination of nucleic acid samples.

Science message board:

micro.nwfsc.noaa.gov/protocols/methods/RNAMethodsDocs/ 10-2002/10.16.02.4177.html

Some reagents used in RNA purification protocols (TRIzol) can absorb at

230nm (also at about 270nm). So, 260/230 gives you an idea of reagent
contamination - which is undesirable for some downstream enzyme
applications, e.g. RT-PCR, cDNA synthesis.

www.biologicalprocedures.com/bpo/arts/1/6/m6.htm

Co-extracted humic acids are the major contaminant when DNA is

extracted from soil. These compounds absorb at 230 nm whereas DNA
absorbs at 260 nm and protein at 280 nm. To evaluate the purity of the
extracted DNA, absorbance ratios at 260 nm/230 nm (DNA / humic acids)
and 260 nm/280 nm (DNA / protein) were determined (see Tables 2 and
3).

Compiled by Hillary Luebbehusen

Internet sources cited

Significance of 260/230 ratio

06/23/04

2
Internet references, continued.

http://www.labora.se/pdf/Detection.pdf

Absorption at 230 nm reflects impurities of e.g. carbohydrates, peptides,

phenols, or aromatic compounds. The 260/230 should be above 2 for
pure samples.

http://appletree.mta.ca/Courses/Biochemistry/BC3531/moleccoursenotes/DNAabsorbance.htm

Absorbance at 230 nm indicates contamination by urea or phenol

(reagents commonly used in nucleic acid isolation).

http://www.brinkmann.com/quant_easy.asp

Absorption at 230 nm reflects contamination of the sample by substances

such as carbohydrates, peptides, phenols or aromatic compounds. In the
case of pure samples, the ratio A260/A230 should be approximately 2.2.

http://www.bio.com/protocolstools/protocol.jhtml?id=p9039

Significant absorbance at 230 nm is indicative of carbohydrate or

polyphenol contamination.

Compiled by Hillary Luebbehusen

Internet sources cited