Mar 02, 2024

Public workspaceCo-extraction of RNA and DNA from animal tissue

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1qrkpgr2/v1
Co-extraction of RNA and DNA from animal tissue
  • Dominik Buchner1
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
Dominik Buchner
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1qrkpgr2/v1
Protocol Citation: Dominik Buchner 2024. Co-extraction of RNA and DNA from animal tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qrkpgr2/v1
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: March 02, 2024
Last Modified: March 02, 2024
Protocol Integer ID: 96045
Keywords: dna from animal tissue sample, dna from animal tissue, animal tissue sample, extraction, rna in the flow, rna, interested in the rna, dna, dna spin, animal tissue, cell debris, remaining protein, tissue, separate tube
Abstract
This protocol describes how to co-extract RNA and DNA from animal tissue samples. Samples are homogenized and simultaneously lyzed by bead-beating. Cell debris is pelleted by centrifugation, the DNA is then subsequently bound to a silica column, while the RNA passes the membrane. The RNA in the flow-through is then precipitated with 70% ethanol and bound to a second silica column. Both, DNA and RNA are washed with different wash buffers to remove remaining proteins and other contaminants and finally eluted in separate tubes. If the user is just interested in the RNA, the DNA spin-column can just be discarded.
Guidelines
Follow general lab etiquette. Wear gloves to prevent contamination of samples. Clean the workspace before starting and after finishing with 80% EtOH.
Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on the supply situation.

Chemicals:
Guanidinium thiocyanate ReagentGuanidinium thiocyanateFisher ScientificCatalog #10503345
Tris ultrapure 99.9% ReagentTris ultrapure 99.9%DiagonalCatalog #A1086.1000
Hydrochloric acid fuming 37% ReagentHydrochloric acid fuming 37%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1003171011
Pre-filter columns ReagentPre Filter Columns - 850 µlBiopolymer Isolation TechnologiesCatalog #MC-01P-100
Guanidinium chloride ReagentGuanidine hydrochlorideFisher ScientificCatalog #10543325
Ethanol absolute ReagentEthanol absolute 99.8%Fisher ScientificCatalog #11994041
Antifoam solution (optional): ReagentSilicon-Antischaumemulsion 30Carl RothCatalog #0734.1

Labware:
2 mL screwcap tubes Reagent2 mL screwcap tubeSarstedtCatalog #72.693
2 mm zirconia beads ReagentZirconia Beads 2 mm diaBioSpec ProductsCatalog #11079124zx
0.1 mm glass beads ReagentGlass Beads 0.1 mm diaBioSpec ProductsCatalog #11079101
EconoSpin mini spin column ReagentEconoSpin mini spin clumn with lidEpoch Life ScienceCatalog #1920-050

Stock solutions:
Amount1 L Tris stock solution Concentration1 Molarity (M) Ph7.5
  • Add Amount121.1 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph7.5 with HCl
  • Adjust volume to Amount1 L with ddH2O

Amount1 L sodium chloride stock solution Concentration5 Molarity (M)
  • Add Amount292.2 g sodium chloride to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph8.5
  • Add Amount121.1 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8.5 with HCl
  • Adjust volume to Amount1 L with ddH2O

Amount1 L DNA wash buffer 2 stock solution Concentration50 millimolar (mM) Tris Ph7.5
  • Add Amount50 mL of Concentration1 Molarity (M) Tris stock solution Ph7.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature


Working solutions:
Amount1 L GITC lysis buffer ( Concentration4 Molarity (M) Guanidinium thiocyanate , Concentration10 millimolar (mM) Tris ) Ph7.5
  • Add Amount472.6 g guanidinium thiocyanate to a beaker
  • Add Amount10 mL of Concentration1 Molarity (M) Tris stock solution Ph7.5
  • Adjust volume to Amount1 L with ddH2O
  • Stir until the GITC is completely dissolved (heating will speed this up)
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L RNA wash buffer 1 ( Concentration900 millimolar (mM) Guanidinium thiocyanate , Concentration10 millimolar (mM) Tris , Concentration20 % (v/v) Ethanol absolute ) Ph7.5
  • Add Amount106.3 g guanidinium thiocyanate to a beaker
  • Add Amount10 mL of Concentration1 Molarity (M) Tris stock solution Ph7.5
  • Add Amount200 mL Ethanol absolute
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L RNA wash buffer 2 ( Concentration100 millimolar (mM) sodium chloride , Concentration10 millimolar (mM) Tris , Concentration80 % (v/v) ethanol absolute ) Ph7.5
  • Add Amount20 mL of Concentration5 Molarity (M) sodium chloride stock solution
  • Add Amount10 mL of Concentration1 Molarity (M) Tris stock solution Ph7.5
  • Adjust volume to Amount200 mL with ddH2O
  • Adjust volume to Amount1 L with ethanol absolute
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L DNA wash buffer 1 ( Concentration2.5 Molarity (M) Guanidinium chloride , Concentration10 millimolar (mM) Tris , Concentration57 % (v/v) Ethanol absolute ) Ph7.5
  • Add Amount238.9 g guanidinium chloride to a beaker
  • Add Amount10 mL of Concentration1 Molarity (M) Tris stock solution Ph7.5
  • Adjust volume to Amount430 mL with ddH2O to dissolve the GuHCl
  • Adjust volume to Amount1 L with Ethanol absolute
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L DNA wash buffer 2 ( Concentration10 millimolar (mM) Tris , Concentration80 % (v/v) ethanol absolute ) Ph7.5
  • Add Amount200 mL DNA wash buffer 2 stock solution to a beaker
  • Adjust volume to Amount1 L with Ethanol absolute
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L elution buffer Concentration10 millimolar (mM) Tris Ph8.5
  • Add Amount10 mL of Concentration1 Molarity (M) Tris stock solution Ph8.5 to a beaker
  • Adjust the volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature
Troubleshooting
Safety warnings
Buffers containing guanidine produce highly reactive compounds when mixed with bleach. Don't mix the extraction waste with bleach or solutions that contain bleach.
Reagents are potentially damaging to the environment. Dispose waste as mandated.
Before start
Make sure all buffers are prepared before starting.
Sample preparation and lysis
5m
For each sample prepare one 2 mL screwcap tube pre-filled with approximately Amount400 mg of 2 mm zirconia beads and 0.1 mm glass beads.
Note
Generally, we just add a small spoon of each type of beads to the tube. As long as the tissue is fully homogenized after bead-beating, the amount of beads is sufficient.

Add up to Amount30 mg of animal tissue to the prepared tube.

Note
For samples with a high RNA content, less starting material might lead to better results. For most sample types Amount10 mg of starting material will yield a sufficient amount of DNA and RNA for downstream analysis.



Add Amount1000 µL GITC lysis buffer to the sample tube.

Note
For complete inactivation and destruction RNAses Amount10 µL of 2-Mercaptoethanol can be added in addition. We usually don't because then the samples have to be handled under a fume hood until all lysate has been handled and discarded appropriately.

If you experience a lot of foam formation after bead-beating consider adding Concentration30 Parts per Million (PPM) silicone antifoam to the lysis buffer when preparing it. See materials for a recommendation.



Immediately bead beat for Duration00:05:00 at maximum speed.

Note
Depending on the bead beater used in this step the time might have to be adjusted. We recommend to bead beat the sample until the material is completely homogenized.



5m
Lysate clearing
10s
CentrifigationRoom temperature, 00:10:00 , at maximum speed

10m
DNA binding
Transfer Amount700 µL of the cleared lysate from step 5the to a silica spin column to bind the DNA in the lysate. Keep the flow-through. Mark the spin column as the DNA column.

Note
The protocol will work with all kinds of silica spin columns. See the materials section for what we use.

If you are only interested in RNA: If only RNA is of interest the DNA spin column can be discarded at this point in the protocol.


RNA precipitation and binding
15s
Add Amount700 µL 70% Ethanol to the flow-through from step 6 to adjust the binding conditions for RNA to bind to the silica column.

Vortex the samples to mix the lysate with the ethanol. Do not centrifuge.
Load the mixture on a second spin column. Mark this column as the RNA spin column.
Centrifigation11000 x g, Room temperature, 00:00:15 and discard the flow-through.

Note
Two loading steps will be necessary to pass the complete volume through the spin column.


15s
Washing steps
15s
Add Amount700 µL RNA wash buffer 1 to the RNA spin column,Centrifigation11000 x g, Room temperature, 00:00:15 and discard the flow-through.
Note
For less experienced users: If you are concerned about needing to much time to process both fractions at the same time and risk RNA degradation it is fine to first finish the RNA extraction until safe storage and then finish the DNA fraction.


15s
Add Amount500 µL RNA wash buffer 2 to the RNA spin column, add Amount500 µL DNA wash buffer 1 to the DNA spin column, Centrifigation11000 x g, Room temperature, 00:00:15 and discard the flow-through.

15s
Add Amount500 µL RNA wash buffer 2 to the RNA spin column, add Amount500 µL DNA wash buffer 2 to the DNA spin column, Centrifigation11000 x g, Room temperature, 00:00:15 and discard the flow-through.

15s
Column drying and elution
4m
Centrifigation11.000 x g, Room temperature, 00:01:00 to dry the silica membrane of the spin columns. Transfer the spin column to a fresh 1.5 mL microcentrifuge tube.
1m
Add Amount100 µL elution buffer directly to the silica membrane. Incubate the column for Duration00:03:00 at TemperatureRoom temperature
Note
The volume of the elution buffer can be adjusted in this step if a higher concentration or higher volume is required for downstream analysis. Usually, every volume in the range from Amount30 µL to Amount200 µL is fine.



3m
Centrifigation11.000 x g, Room temperature, 00:01:00 , store the eluted RNA at Temperature-80 °C and the eluted DNA at Temperature-20 °C

Expected result

The described protocol was tested with different kinds of invertebrate samples, we expect it to work with all animal tissue.
Top row: Plecoptera sample and two Trichoptera samples.
Lower row: Three Gammarus samples.

28S/18S bands are clearly visible and should have a clear band. Genomic DNA is free from RNA contamination. There is some DNA contamination in the RNA extracts. If DNA-free RNA is needed for downstream analysis consider treating the RNA samples with DNase and cleaning them up with an RNA cleanup protocol afterward (see RNA Cleanup with magnetic beads).




1m