The DNase step is one of the most common causes of degradation or loss of the RNA during your extraction. DNase digestion is frequently performed on the spin column and although this can be a great way to save time on the post extraction processing, it is not an efficient method for samples with large amounts of DNA (for example, spleen, thymus, and even some soils). In these cases, DNase digestion in solution is necessary.
The typical protocols for DNase involve inactivation of the enzyme using EDTA and heat. Both of these things can cause problems in RT-PCR. EDTA can inhibit the RT-PCR enzymes and heating the RNA can cause a reduction in integrity. Additionally, most DNase enzymes are stored frozen and need to be aliquoted to avoid freeze/thaw cycles that can reduce enzyme efficiency.
We came up with a better system that protects the RNA all the way to the final step: DNase Max.
DNase Max is a liquid room temperature stable DNase with very high activity (1 ul of enzyme can digest 30 ug of DNA in 20 minutes). The best part is the clean up step. The DNase comes with a highly specific removal resin that binds the enzyme and cations and pulls them out of the RNA sample making it ready to use in qRT-PCR without any inhibitory additives or heat steps. The resin is so efficient that completely removes 10 units of enzyme in the reaction, (figure below, lanes 3-4) compared to an alternative resin method incapable of removing the just 2 units of DNase enzyme used (lanes 1-2).
This means that you can protect your precious RNA as well as hours of work invested and get better accuracy in gene expression assays.
DNase MaxRemoval Resin completely removes DNase. Samples were subjected to DNase treatment and enzyme removal using the DNase Max™ Kit or a competitor’s kit, and then analyzed for residual DNase activity using the MO BIO DNase-free certification assay. Lane 5 is the negative control and did not receive DNase. Samples were incubated for 1 hour at 37C, followed by inactivation for 5 minutes at 65C. Results are shown on a 1% agarose gel. The DNase Max Removal Resin successfully removed the DNase (lanes 3-4), while the competitor’s resin failed to remove all of the DNase from the samples (lanes 1-2).
Other tools to protect your RNA.
Isolation of RNA, no matter what the source, is nerve wracking, but especially when samples are limited or irreplaceable. Because RNA is so labile, working quickly but carefully is the key. There are ways to protect your RNA during the procedure so that you can work at a relaxed pace and without so much anxiety.
The use of BME, consistent and fast homogenization, and RNase-free DNase with removal resin will be your ticket to success in every prep no matter what the sample. Certified RNase-free gloves are a great extra to have as well as UltraClean Lab Cleaner for removal of nucleases from the bench and equipment. We use these routinely in our labs.