FAQ: Does the kit work with frozen tissue, tissue stored in preservatives (ethanol, DMSO-salt/DESS, or RNAlater®), FFPE samples, or OCT-preserved tissues?

We have successfully tested several of the options for preservation of frozen tissue samples, but fresh input material is always best whenever possible. When fresh samples are not available, freezing is the recommended alternative, ideally shock freezing with liquid nitrogen and storing at -80°C. When thawing, the goal is not to give the nucleases much time to harm the DNA, and you can ensure this by carrying out the homogenization rapidly and mixing with the lysis buffer immediately after the sample is thawed and homogenized. Interestingly, frozen tissue samples often give better results in terms of DNA fragment length and integrity when compared to those stored in preservation reagents. We have tested Monarch DNA/RNA Protection Reagent (NEB #T2011), RNAlater, DNA/RNA Shield, and RNAssist. Although these reagents effectively preserve the samples, their presence prevents rapid digestion of the tissue material during the initial phase of lysis, which seems to result in some shearing/DNA damage. In our experience, the Monarch DNA/RNA Protection Reagent provides somewhat better results than RNAlater and RNAssist.

Ethanol-preserved tissue can be used as input material. Before starting the lysis procedure, ethanol should be removed by washing samples with a medium-salt buffer; further details are available on request (contact us at info@neb.com).

DMSO-preserved frozen cells have been tested and gave good quality DNA. We have not tested DESS but presume that it should work as well.

FFPE samples are not suitable for HMW DNA isolation as they are highly crosslinked, resulting in significant fracturing and denaturing of the DNA during the stringent purification workflow that is required for these samples. Typically, FFPE samples are not a target input material for those seeking HMW DNA, since purified DNA will mostly be < 1 kb and will be denatured.

OCT samples may potentially work, as the OCT fixed samples are not crosslinked, but we have not tested this. We encourage users to give it a try using our free trial kits. It is always good to stay in the middle range of the recommended input amount (e.g., 10-15 mg) to start. Before starting the DNA extraction, remove as much OCT as possible.

  1. Monarch-HMW-Video_Tissue_1920

    Monarch HMW DNA Extraction from Tissue: Protocol Overview

    In this video, we walk through the protocol for extraction of high molecular weight DNA (HMW DNA) from tissues.