May 14, 2020
DNA and RNA stabilization
- 1University of Manitoba
Protocol Citation: Neilier Junior 2020. DNA and RNA stabilization. protocols.io https://dx.doi.org/10.17504/protocols.io.be2pjgdn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 13, 2020
Last Modified: May 14, 2020
Protocol Integer ID: 35631
Keywords: DNA, RNA, RNA stabilization, Molecular Biology, Biochemistry, RNA extraction, RNase free, Nucleic acids, RNA-seq,
Abstract
DNA and RNA stabilization solution is an aqueous and non-toxic tissue storage reagent that rapidly permeates tissues to stabilize and protect nucleic acids in fresh specimens. Tissue pieces are harvested and immediately submerged in stabilization solution for storage. It can be used for DNA and RNA preservation with most tissues, cultured cells and is compatible with most DNA/RNA isolation methods. Proteins are precipitated, but can still be used for analyzes that do not require their native structure, such as western blotting or gel electrophoresis. Samples in DNA and RNA stabilization solution can be stored indefinitely in RNAlater Solution at –20 °C or below.
Guidelines
Use only RNase-free tubes, tips and flasks.
Materials
MATERIALS
Ammonium SulfateP212121
Sulfuric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #258105
Sodium CitrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #71402
EDTAThermo FisherCatalog #17892
Use only RNase-free tubes, tips and flasks.
Safety warnings
Wear personal protective equipment: gloves, clothing and mask.
Before start
Organize your workspace.
Make sure all solutions and equipment are available. Plan the experiment!
Stabilization solution preparation (1.5 L)
Stabilization solution preparation (1.5 L)
- 935 mL of autoclaved MilliQ water
- 700 g ammonium sulfate
- Stir until dissolved
- Add 25 mL of 1 M sodium citrate
- Add 40 mL of 0.5 M EDTA
- Adjust to pH 5.2 using concentrated H2SO4
- Complete the volume to 1.5 L with autoclaved MilliQ water
Note
- Store at room temperature.
- If any precipitation of stabilization solution is seen: heat to 37 °C and agitate to redissolve it.
Procedure
Procedure
- Do not freeze tissues before immersion in DNA and RNA stabilization solution.
- Use stabilization solution with fresh tissue only.
Add the stabilization solution in an appropriate 1.5 mL microtube.
Note
Use the minimum volume ratio 1:5 (tissue : solution).
Cut large tissue samples to approx. 3 mm in any single dimension and transfer to microtube containing the solution.
Store at 4 °C overnight to allow the solution to thoroughly penetrate the tissue.
Do not freeze samples in stabilization solution immediately.
Important notes
Important notes
The DNase and RNase inactivation is reversible. So, do not rinse stabilization solution from samples before DNA and/or RNA extraction.
Tissue homogenization should be rapid once the tissue is in lysis/denaturation solution to nucleic acids extraction.
Remove the stabilization solution from the cells prior to extraction for isolating DNA and RNA from cells.
With the aid of a pipette, remove excess ofstabilization solution from the tube and then use an absorbent lab wipe or paper towel to help remove as much of the stabilization solution as possible.