DNA/RNA isolation considerations when using Illumina library prep kits

A wide variety of DNA and RNA sample types and extraction methods can introduce inhibitors to enzymatic reactions. Inhibitors, if not properly removed, can negatively affect many types of enzymatic reactions including reverse transcription, end repair, A-Tailing, adapter ligation, and PCR. This bulletin outlines important considerations for isolation and purification of DNA and RNA before library preparation with Illumina library preparation kits.

Which inhibitors are of concern?

Some inhibitors, or contaminants, are inherent to sample type or sample source, such as hemoglobin in blood or humic/fulvic acid in plant samples. Others are introduced through the sample treatment or extraction method such as EDTA, heparin, or phenol:chloroform.

Inhibitors can prevent the enzyme from binding to the target substrate (i.e., proteins coating DNA/RNA and thus preventing enzyme binding), reduce enzyme function, or degrade the enzyme itself (e.g., proteinases, detergents, phenol, and pH).

What if I suspect or detect contaminants in my DNA/RNA samples?

The best way to prevent enzyme inhibition or impaired performance is to couple careful sample handling with extraction protocols optimized to efficiently purify inhibitor-free nucleic acids. The final pH of the sample after eluting or resuspending nucleic acids should be within a range of 7.0-8.5.

Illumina recommends UV spectrophotometry for purity assessment, and fluorometric based methods such as Qubit or Pico/RiboGreen for nucleic acid quantitation. The most common method to assess the purity of nucleic acids in solution is to measure the 260/280 and 260/230 ratios by UV spectrophotometry:

Which inhibitors are found in different sample types?

Which inhibitors are found in different samples treatments/extraction methods?

How to remove contaminants from DNA/RNA samples?

If the sample has contaminants that affect downstream enzymatic reactions, additional purification steps such as a filter-based spin column repurification may help in the removal of contaminants and/or concentration of the sample.

Note that this list does not include all possible inhibitors of enzymatic reactions, but rather the most common contaminants that originate from sample source or sample treatment/extraction method.

For any feedback or questions regarding this article (Illumina Knowledge Article #1249), contact Illumina Technical Support [e-mail].