Purity - Does my sample contain contaminants?

Nucleic acid samples can become contaminated by other molecules with which they were co-extracted and eluted during the purification process or by chemicals from upstream applications.

Purification methods involving phenol extraction, ethanol precipitation or salting-out may not completely remove all contaminants or chemicals from the final eluates. The resulting impurities can significantly decrease the sensitivity and efficiency of your downstream enzymatic reactions.

Along with identifying common chemical contaminants, differentiating between different types of nucleic acids is also important when assessing sample purity. For example, large amounts of unwanted RNA in a DNA template sample can result in overestimation of DNA concentration and reduce the yields of downstream PCR assays.

Assessing the purity of nucleic acid samples helps you make informed decisions regarding whether a sample is suitable for processing in the next analysis step or whether it should be discarded from the workflow or reprocessed before further analysis.

The purity of a sample greatly impacts the success of downstream analyses. The presence of inhibitors and proteins in purified nucleic acids can inhibit enzymatic reactions such as restriction enzyme digestion, PCR and ligation, as well as chemical reactions like colorimetric assays. Furthermore, residual genomic DNA in RNA samples can influence the CT values of qPCR results. Large amounts of RNA present in DNA samples can result in chelation of Mg2+, reducing PCR yields.

UV spectrophotemetry measurements enable calculation of nucleic acid concentrations based on the sample's absorbance at 260 nm. The absorbances at 280 nm and 230 nm can be used to assess the level of contaminating proteins or chemicals, respectively. The absorbance ratio of nucleic acids to contaminants provides an estimation of the sample purity, and this number can be used as acceptance criteria for inclusion or exclusion of samples in downstream applications.

However, the disadvantage of this method is that classical spectrophotometers cannot differentiate between types of nucleic acids. The calculation for determining concentration relies only on the absorbance at 260 nm, but both DNA and RNA in the sample will contribute to the absorbance value. In reality, the method actually measures all nucleic acids in the sample and can result in overestimation of the RNA or DNA concentration.

Furthermore, A260/A230 ratios can be misleading when other UV-absorbing molecules are in the elution buffer. Measuring blank samples and subtracting the background prevents these molecules from interfering with the absorbance readings, but their presence in the sample still affects downstream applications. Using a fluorescent dye-based method can help quantify only the molecule of interest, but such a method is unable to provide information regarding contamination.

QIAGEN’s next-generation spectrophotometer combines the advantages of both methods. By applying smart analysis algorithms, the QIAxpert system unmixes the spectra and fits reference sample and buffer components to correctly discriminate between DNA, RNA and impurities.

Table 1. Assessment of nucleic acid quality control parameters by various technologies

Spectral content profiling analysis with the QIAxpert system goes one step further than classical methods for purity testing, measuring a broader light spectrum and applying smart analysis algorithms. Specifically quantifying DNA, RNA or total nucleic acids along with other residues and impurities gives you full insight into your sample quality.