Protect your RNA samples during DNase treatment

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One of the most common causes of degradation or loss of RNA during extraction is the DNase step. DNase digestion is frequently performed on a spin column. Although this can be a great way to save time on 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.

Typical protocols for DNase digestion involve inactivation of the enzyme using EDTA and heat. These methods can cause problems in downstream applications such as RT-PCR. EDTA can inhibit the RT-PCR enzymes and heating 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.

A better solution that protects RNA – DNase Max

We came up with a better solution. DNase Max is a room temperature stable liquid that includes high-activity DNase I. This proprietary enzyme removes up to 30 µg of DNA in 20 minutes. The best part is the cleanup. DNase Max comes with a highly specific removal resin that efficiently binds the enzyme and cations, pulling them out of the RNA sample making it ready for use in downstream PCR applications without any inhibitory additives or heating steps. This means that precious RNA can be protected and hours of work can be saved, resulting in better accuracy in gene expression assays.

Figure 1. DNase Max removal 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 QIAGEN Services DNase-free certification assay. Lane 5 is the negative control and did not receive DNase. Samples were incubated for 1 hour at 37ºC, followed by inactivation for 5 minutes at 65ºC. 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 the DNase from the samples (lanes 1–2), resulting in degradation.

Figure 1. DNase Max removal 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 QIAGEN Services DNase-free certification assay. Lane 5 is the negative control and did not receive DNase. Samples were incubated for 1 hour at 37ºC, followed by inactivation for 5 minutes at 65ºC. 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 the DNase from the samples (lanes 1–2), resulting in degradation.

 

Other tools to protect your RNA

Isolation of RNA, no matter what the source, is nerve wracking especially when samples are limited or irreplaceable. Because RNA is so labile, working quickly but carefully is the key. There are additional ways to protect your RNA during the procedure that make RNA isolation easier. Using β-Mercaptoethanol as well as consistent and fast homogenization can also improve RNA isolation. In addition, using the DNase Max Kit with removal resin can make RNA isolation successful in every prep no matter what the sample.

 

Authorship note: This article was originally published by MO BIO and has been edited by Heather Martinez.

This article was compiled from the contributions of multiple authors. Please see the end of the post for details.

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