Bead based sample cleanups (e.g., Ampure XP, RNAClean XP) and spin column-based protocols (e.g., Qiagen, Zymo, NorgenBiotek) tend to be the most efficient ways to remove chemical contaminants. For genomic DNA samples to be sequenced on Illumina sequencers, we suggest spin columns since DNA treated this way will always dissolve well and completely.
Please test for chemical contamination by spectrophotometry (e.g., Nanodrop), concentrations should be measured by fluorometry instead (Qubit, Quantus, plate reader, …) :
- Please see this guide from the University of Arizona on the interpretation of Nanodrop data. Skewed absorption ratios indicate that there is chemical contamination, but not precisely which contaminant and if it will be deleterious or not,
- The 260/230 nm and 260/280 nm absorption ratio measurements are most frequently used to assess purity. Please see the sample requirements page for the recommended values for your protocol. However, it is certainly helpful to also record the entire UV absorption spectrum as it provides additional information. For RNA the 260/230nm ratio should be >1.5 and the 260/280nm ratio 1.8-2.1; For DNA the 260/230nm ratio should be >2 and the 260/280nm ratio 1.8-2.0 .
- In case the absorption ratios are skewed, it is often worth checking if any alcohol was carried over from the spin column or bead washes. Any organic substance, including ethanol, will skew the 260/230 nm ratios. One can vent the open sample tube (for example for 20 minutes) on the lab bench and measure again afterwards to see if the contamination has disappeared.
- The spectrophotometer ratios themselves become easily misleading at very low DNA or RNA concentrations (10 ng/ul or less). In these cases the nucleic acid samples contribute very little to the signal and the slightest contamination dominates the readings. Please record the absorption spectra.
Multiple protocols are available to remove DNA or RNA contaminants. Please find our suggestions for affordable solutions for Illumina sequencing below.
RNA samples need to be DNA-free. The RNA isolation protocol should always include a DNase digestion step; in problematic cases use RNA-clean & concentrator kits with DNase. On an agarose gel, DNA contamination will be visible as a smear or band of fragments considerably larger than the RNA (>10 kb). On Bioanalyzer RNA-chips, DNA contamination will be visible in the size range 4 kb to 10 kb.
If you are using a Trizol protocol for the RNA extractions we would highly recommend cleaning the samples afterwards with a spin column kit (e.g. RNA-clean & concentrator kits) to remove any phenol traces.
Please note that the additional column cleanup is mandatory for RNA samples isolated from blood PAXgene or Tempus tubes (for blood sample preservation) or with the accompanying PAXgene and Tempus RNA isolation kits.
DNA samples need to be RNA-free. The DNA isolation protocol should always include an RNase digestion step; in problematic cases we recommend using RNase I (e.g. add 1 ul RNAse I to your sample and incubate at 30 degrees C for 20 minutes). RNase I does not require a special buffer (it works in TE buffer) and can be completely inactivated by heating at 70°C for 15 minutes. Thus, a removal of the enzyme and of a buffer can be avoided in many cases. (For some applications small amounts of RNase I can remain in the sample without inactivation or removal — please inquire). For the removal of RNase I, Ampure XP beads (or similar) or DNA-clean & concentrator kits will work fine (we suggest extending incubation times for elutions from the columns to at least 5 minutes or to perform two elutions). DNA samples can be QC-ed easily by agarose gel electrophoresis and ethidium bromide staining. The stain will make both DNA and RNA visible. RNA will run as an halo-like smear in the range 50 to 200 bp.
For the removal of chemical contaminants solid-phase paramagnetic bead cleanups (SPRI-beads) are a solution suitable for high-throughput processing. The first such products were Ampure XP (for DNA) and RNAClean XP from Agencourt/Beckman. Many companies are now selling lower-cost versions, for example MagBioGenomics DNA beads and RNA beads.
We can recommend this EdgeBio magnetic plate for bead cleanups in 96-well plates.
- For spin-column cleanups: Please perform the optional steps described in the manual. Always perform at least two spin column washes (with the kit wash buffer) after binding of the sample to the column matrix. Also, add a short “dry spin” of the column after the washes and before the elution buffer addition to avoid any carryover of the ethanol wash buffer.
- We suggest extending incubation times for elutions of DNA samples from spin columns to at least 5 minutes – or to perform two consecutive elutions instead.
- NEVER use heparin as an anticoagulant for blood samples destined for DNA or RNA sequencing. EDTA (preferred) or citrate anticoagulants should be used. Heparin co-purifies with nucleic acids and inhibits multiple types of enzymes like polymerases and ligases.
- Avoid using glycogen as co-precipitant.