Quantification of nucleic acids

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There exist different methods to establish the concentration of a solution of nucleic acids (RNA and double or single-stranded DNA) . One approach to determining DNA concentrations is to load part of the solution on an agarose gel, stain the bands with ethidium bromide, and compare the intensity of the stained bands with that of marker bands of known mass. This method is only semi-quantitative but adequate for some purposes. For many applications involving PCR, however, greater accuracy is required, and spectrophotometric or fluorometric methods of DNA quantification should be used.

[edit] Spectrophotometric quantification of nucleic acids

Due to their molecular structures, both DNA and RNA absorb UV light of 250 to 270nm wavelength, with a maximum at 260nm. The reading at 260nm allows calculation of the concentration of nucleic acid in the sample, as there exists a linear relationship exists between the absorption of light and the nucleic acid concentration.

For this kind of measurements, a spectrophotometer is used. Inside a spectrophotometer, the sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample.

Using the Beer-Lambert law it is possible to relate the amount of light absorved and the concentration of the absorbing molecule. This way, an OD of 1 corresponds to 50 µg/ml for double-stranded DNA, 37 µg/ml for single-stranded DNA and 40 µg/ml for RNA.

Furthermore, spectrophotometric measurement gives an idea of the purity of the sample. The ratio of absorbance at 260 nm to absorbance at 280 nm is used to indicate contamination by proteins or phenol. An A260/A280 ratio greater than 1.9 for DNA, or greater than 1.8 for RNA is indicative of samples that are free of these contaminants. Absortion at 230 nm reflects impurities such as carbohydrates, peptides, urea or aromatic compounds. For pure samples, the ratio should be above 2.

[edit] Purity and Contamination Problems

• 260/280 should be 1.8 for DNA and 2.1 for RNA. Blow ratio might be protein contamination.
• 260/230 should be the same as the 260/230. if the value is lower it may be due to phenol and other aromatic compounds, urea, carbohydrates and amino acids, humic acid, etc.
• A310 or A340 should be zero. This is an indicator of contamination.
• Dust in the sample results in impossible values.
• In the case of negative values the solution used as blank should be measured again to see if was blanked incorrectly, alternatively in could be due to fluorescence of a dye.
• RiboGreen® or is used when the concentration of RNA is low (0-0.5 units @A260)

[edit] purification kits

There are two most common purification methods: phase separation and column.

The exact composition of Trizol (invitrogen) or TriReagent (MRC) is confidential. The original protocol contained a 1mL solution of (4M guanidium thiocyanate, 25nM sodium citrate 0.5% N-Laurosylsarcosine and 0.1 b-mercaptoethanol) mixed with 0.1mL 2M sodium acetate and 1mL acid phenol and 0.2mL chloroform/isoamyl alcohol (49:1) or 0.1mL bromocholoropropane. Phenol is the main ingredient and the major contaminant when assessing purity, therefore is is essential not to disturb the organic phase (phenol rich).

The qiagen buffers on the other hand do not contain phenol; the lysis buffer RTL has a very sharp and strong 260 peak. Whereas Qiagen’s Elution Buffer is TE pH 8.0.

[edit] Blank

H2O, EtOH, iPrOH, TE and other buffers when used as a blank are identical to water. So blanking H2O or TE makes no difference. Instead buffers have profound effects due to pH and ionic composition which change the spectrum of nucleic acids. A280 increases with pH. Low pHs such as DEPC H2O (5-6) result in a lowered 260/280. TE pH 8.0 is recommended for nucleic acids.

[edit] Ethanol

EtOH and iPrOH make RNA and DNA hard to solubilize. when the nucleic acids form a pellet or small invisible ones the values are very low or off the charts. When the RNA is fully solubilized though EtOH result only in an increased 280. (no negative values, same 230, same 260. a bit shifted). This probably is due to the fact ethanol is pH 10.

[edit] References

This article or section may contain inappropriate or misinterpreted citations which do not verify the text. Please help improve this article by checking for inaccuracies. This article has been tagged since March 2007. (help, talk, get involved!)

• Trizol: the single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty something years on, Nature protocols ?

• Ribogreen [1]

• Ambion guide [2]

• GE Healthcare guide [3]

• The significance of 260/230 [4]