Apr 09, 2026
Manual cryogenic pulverisation of formalin-fixed or ethanol-preserved tissue for RNA extractions
- Ashleigh Porter1
- 1CSIRO
Protocol Citation: Ashleigh Porter 2026. Manual cryogenic pulverisation of formalin-fixed or ethanol-preserved tissue for RNA extractions. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx8qndv8j/v1
Manuscript citation:
Porter, Ashleigh F. Manual cryogenic pulverisation of formalin-fixed or ethanol-preserved tissue for RNA extractions.
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: February 24, 2026
Last Modified: April 09, 2026
Protocol Integer ID: 243908
Keywords: manual cryogenic pulverisation of museum specimen tissue, manual cryogenic pulverisation of formalin, tissue for rna extraction, manual cryogenic pulverisation, rna extraction, use in nucleic acid extraction, such as rna extraction, nucleic acid extraction, museum specimen tissue, preserved tissue, rna yield, extraction, rna, storage conditions of the museum specimen, fixed tissue, ethanol, process of flash freezing, museum specimen, read sequencing, long read sequencing
Abstract
A method describing the manual cryogenic pulverisation of museum specimen tissue (formalin-fixed or ethanol-preserved) for use in nucleic acid extractions, such as RNA extractions. Fixed tissue is pulverised by the process of flash freezing and sudden impact, which creates a "dust" that undergoes a hot proteinase K digest before extraction.
RNA yield is expected to be related to the age, collection and storage conditions of the museum specimen. RNA will be fragmented and not suitable for long-read sequencing.
Attachments
Guidelines
For best results, apply historical DNA laboratory methods and practices for this method.
Materials
- historical DNA/RNA bench or lab space
- liquid nitrogen
- cryogenic gloves
- eppendorf tubes
- biopulveriser
- metal tray
- tongs
- fine forceps
- spatula
- bleach and ethanol, for cleaning
- proteinase K
- heat block/shaker
- 2 X buffer
- RNA extraction kit
Safety warnings
Wear PPE (gloves, laboratory coat, goggles) when handling specimens that have been fixed in formalin/formaldehyde and handle specimens in a chemical hood if possible.
Wear PPE (gloves, laboratory coat, goggles) when handling specimens that have been preserved in EtOH and handle specimens in a chemical hood if possible.
Wear PPE (gloves, laboratory coat, goggles) when handling bleach and ethanol.
Wear PPE (cryogenic gloves, laboratory coat, goggles) when handling liquid nitrogen and any equipment that has been bathed in liquid nitrogen.
Practice caution when using the biopulveriser.
Ethics statement
The use of museum tissue in this method requires approval from individual Institutional Ethics Board or equivalent ethics committee.
Cryogenic pulverisation of museum tissue
Cut a lentil sized piece of formalin-fixed, ethanol-preserved tissue, or otherwise chemically preserved tissue
Weigh tissue and record specimen, weight and tissue origin (e.g., organ)
Store tissue in eppendorf tube, in fluid that it was originally preserved in (or 70% ethanol) so that it is fully submerged and will not dry out.
Prepare liquid nitrogen and bring to historical laboratory, along with tissue tubes, biopulveriser, proteinase K and 2 X buffer.
Prepare eppendorf tubes containing 20uL proteinase K and 200uL 2 X Buffer for the number of samples that you will process.
Remove the 70% ethanol or other preservative from your tissues with a pipette.
Pour ~0.5L of liquid nitrogen into cryogenically safe transport container to use per 10 samples.
Pour liquid nitrogen into a cryogenically safe container for bathing the biopulveriser (e.g., metal tray, dimensions 20 cm x 20 cm x 10 cm) so that there is a shallow layer of liquid nitrogen. Place biopulveriser into liquid nitrogen so that each part is bathed by liquid nitrogen for ~1 minute (Diagram, 1.). Use tongs and gloves to avoid cryogenic burns.
Remove biopulverser components and place on clean bench and fit pieces 1 and 2 together (Diagram, 2.). With clean fine forceps, take the lentil sized piece of tissue and place it on the end of piece 3 (usually the rough edge) (Diagram, 3.). Using a clean eppendorf, carefully load the biopulveriser container with ~0.5-1ml fresh liquid nitrogen (Diagram, 4.). Wait until the liquid nitrogen has "dried off" in the container.
Place piece 3 into the biopulversier (Diagram, 5.). Using the hammer, crush the tissue with a hard blow (once or twice) (Diagram, 6.) so that the tissue is entirely pulverised (it should resemble dust). Using a clean spatula, scrape the tissue dust into the pre-prepared eppendorf with proteinase K and 2 x buffer (Diagram, 7.).
Repeat for all samples, cleaning the tweezers, spatula, and biopulveriser parts between uses with bleach, water, and ethanol.
Protein digest
Place the eppendorf tubes containing the pulverised tissue, proteinase K and 2 x Buffer into a pre-heated shaker (65
RNA extraction
Using the Zymo Quick-RNA Mini Prep kit, extract RNA using manufacturers instructions (with DNaseI treatment). Use a 40uL elution at the final step to increase RNA yield.
Acknowledgements
CSIRO CERC Fellowship