Sep 19, 2023
Version 2
Co-extraction of RNA and DNA from soil and sediment samples V.2
- 1University of Duisburg-Essen, Aquatic Ecosystem Research;
- 2University of Duisburg-Essen
Protocol Citation: Dominik Buchner, Lisa Wolany 2023. Co-extraction of RNA and DNA from soil and sediment samples. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb6m4vx1/v2Version created by Dominik Buchner
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: September 19, 2023
Last Modified: September 19, 2023
Protocol Integer ID: 88003
Keywords: rneasy powersoil dna elution kit, rneasy powersoil total rna kit, extracting rna, sediment samples this protocol, dna from soil, sediment sample, rna, extraction, purelink hipure plasmid miniprep kit, sediment, dna, components of the kit, hipure columns from invitrogen, sample, soil, kit
Abstract
This protocol describes extracting RNA and DNA from soil and sediment samples. 2 g of soil or up to 5 g of sediment can be processed in one extraction. The protocol is based on the RNeasy PowerSoil Total RNA Kit but does not rely on any components of the kit. It also replaces the RNeasy PowerSoil DNA Elution kit. The JetStar 2.0 columns are replaced with the HiPure Columns from Invitrogen's PureLink HiPure Plasmid Miniprep Kit. A lot of the buffers can be found in the following patent https://patents.google.com/patent/US7459548B2/en.
Guidelines
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.
Consider working with two layers of gloves when working with phenol and change gloves often.
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.
As an alternative to the PureLink Nucleic Acid Purification Rack from Thermo, we designed a 3D printable rack including a waste container. Both parts form the rack that can be used for purification, reducing the needed 15 mL tubes and corresponding waste immensely.
3D printed rack for up to 24 columns
Column_Rack_PureLink_HiPure_Druck.stl377.9KB Column Rack PureLink HiPure Bottom.stl1.4KB Chemicals:
Sodium phosphate dibasic Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0876-100G
Guanidinium thiocyanate Guanidinium thiocyanateFisher ScientificCatalog #10503345
Sodium phosphate monobasic Sodium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0751-100G
SDS ultrapure Sodium dodecyl sulfateDiagonalCatalog #A1112.0500
Sodium chloride Sodium chlorideFisher ScientificCatalog #10616082
Tris ultrapure 99.9% Tris ultrapure 99.9%DiagonalCatalog #A1086.1000
Hydrochloric acid fuming 37% Hydrochloric acid fuming 37%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1003171011
Aluminium ammonium sulfate dodecahydrate alAluminium ammonium sulfate dodecahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2140-500G
Phenol/chloroform/isoamyl alcohol 8 ROTI Phenol/Chloroform/Isoamyl alcoholCarl RothCatalog #A156.3
Tri-Sodium citrate tri-Sodium citrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #1110371000
Citric acid Citric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #251275-100G
MOPS MOPSCarl RothCatalog #6979.3
Sodium hydroxide Sodium Hydroxide PelletsFisher ScientificCatalog #S318
PureLink HiPure Plasmid-MiniPrep-Kit PureLink HiPure Plasmid-MiniPrep-KitThermo Fisher ScientificCatalog #K210002
Labware:
15 mL centrifuge tubes, Ultra-High PerformanceCentrifuge tubes Ultra-High PerformanceVWR International (Avantor)Catalog #525-1091
0.1 mm glass beads Glass Beads 0.1 mm diaBioSpec ProductsCatalog #11079101
0.5 mm glass beads Glass Beads 0.5 mm diaBioSpec ProductsCatalog #11079105
1 mm zirconia/silica beads Zirconia/Silica Beads 1 mm diaBioSpec ProductsCatalog #11079110z
2 mm zirconia beads Zirconia Beads 2 mm diaBioSpec ProductsCatalog #11079124zx
Stock solutions:
1 L SDS stock solution 10 Mass / % volume
- Add 100 g SDS ultrapure to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L sodium chloride stock solution 5 Molarity (M)
- Add 292.2 g sodium chloride to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 8
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 8.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8.5 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL trisodium citrate stock solution 300 millimolar (mM) 5
- Add 38.7 g tri-Sodium citrate to a beaker
- Adjust pH to 5 with citric acid
- Sterilize by filtering and store at Room temperature
500 mL MOPS stock solution
- Add 104.64 g MOPS to a beaker
- Adjust volume to 450 mL with ddH2O
- Adjust pH to 7 with sodium hydroxide
- Sterilize by filtering and store at Room temperature
Working solutions:
500 mL bead-beating solution (180 millimolar (mM) sodium phosphate , 120 millimolar (mM) guanidinium thiocyanate ) 8
- Add 12.8 g sodium phosphate dibasic to a beaker
- Add 7.1 g guanidinium thiocyanate
- Adjust volume to 490 mL with ddH2O
- Adjust pH to 8 by adding sodium phosphate monobasic
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL lysis solution (150 millimolar (mM) sodium chloride , 4 Mass / % volume SDS , 500 millimolar (mM) Tris ) 8
- Add 200 mL of 10 Mass / % volume SDS stock solution
- Add 15 mL of 5 Molarity (M) sodium chloride stock solution
- Add 250 mL of 1 Molarity (M) Tris stock solution 8
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL inhibitor removal solution (120 millimolar (mM) aluminium ammonium sulfate dodecahydrate )
- Add 27.2 g aluminium ammonium suldate dodecahydrate to a beaker
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL precipitation solution (5 Molarity (M) sodium chloride , 30 millimolar (mM) sodium citrate ) 5
- Add 146.1 g sodium chloride to a beaker
- Add 50 mL of 300 millimolar (mM) tri-Sodium citrate stock solution 5
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL column equlibration buffer (500 millimolar (mM) sodium chloride , 50 millimolar (mM) MOPS , 15 % (v/v) isopropanol ) 7
- Add 50 mL of 5 Molarity (M) sodium chloride stock solution to a beaker
- Add 25 mL of 1 Molarity (M) MOPS stock solution 7
- Add 75 mL isopropanol
- Sterilize by filtering and store at Room temperature
500 mL RNA elution buffer (750 millimolar (mM) sodium chloride , 50 millimolar (mM) MOPS , 15 % (v/v) isopropanol ) 7
- Add 75 mL of 5 Molarity (M) sodium chloride stock solution to a beaker
- Add 25 mL of 1 Molarity (M) MOPS stock solution 7
- Add 75 mL isopropanol
- Sterilize by filtering and store at Room temperature
500 mL DNA elution buffer (1250 millimolar (mM) sodium chloride , 50 millimolar (mM) Tris , 15 % (v/v) isopropanol ) 8.5
- Add 125 mL of 5 Molarity (M) sodium chloride stock solution to a beaker
- Add 25 mL of 1 Molarity (M) Tris stock solution 8.5
- Add 75 mL isopropanol
- Sterilize by filtering and store at Room temperature
1 L elution buffer (10 millimolar (mM) Tris ) 8.5
- Add 10 mL Tris stock solution 8.5 to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
Safety warnings
Phenol/chloroform/isoamyl alcohol is highly corrosive and a carcinogen. Do not inhale vapors, perform all steps under a fume hood, andcontrol your gloves regularly for spills. Store the phenol/chloroform/isoamyl alcohol in a cool and dry space.
Always have PEG 400 ready when working with phenol to absorb small spills. Check the SDS before starting to work with any chemicals.
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.
Cell lysis
42m
Prepare one 15 mL conical centrifuge tube per sample by adding 0.5 g of a) 0.1 mm glass beads, b) 0.5 mm glass beads, c) 1 mm zirconia/silica beads d) 2 mm zirconia beads.
Note
Use high-quality centrifuge tubes for the bead-beating process and phenol/chloroform handling. See the materials section for a recommendation. Low-quality tubes may leak and contaminate the centrifuge or fume hood with phenol.
5m
Add up to 2 g of soil or up to 5 g of the sediment sample.
5m
Add 2.5 mL bead-beating solution , 0.25 mL lysis solution , 0.8 mL inhibitor removal solution and 3.5 mL phenol/chloroform/isoamyl alcohol 8 . Two layers will appear in the centrifuge tube.
5m
Control that the cap is closed properly. Vortex until the biphasic layer disappears.
Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 00:15:00 .
15m
2500 x g, Room temperature, 00:10:00
10m
Transfer 3.5 mL of the upper aqueous phase to a new 15 mL centrifuge tube. Make sure not to pierce the biphasic layer and to not transfer any liquid from the lower phenol phase. If this happens by accident, recentrifuge the sample and repeat. In soil high in organic matter the biphasic layer will be much thicker than in sediment samples.
Note
If you a 45° centrifuge rotor is used the biphasic layer will also be shifted by 45°.
From this point on we use regular centrifuge tubes and have not encountered any issues by doing so.
2m
Inhibitor removal
30m
Add 1.5 mL precipitation solution . The mixture will be cloudy at first but will clear up after vortexing for a few seconds.
5m
Incubate at 4 °C for 00:10:00 .
10m
2500 x g, Room temperature, 00:10:00
10m
Without disturbing the pellet (if there is one), transfer the supernatant to a new 15 mL centrifuge tube.
5m
Nucleic acid precipitation
1h 5m
Add 5 mL isopropanol. Mix by vortexing and incubate at Room temperature for 00:30:00 .
30m
2500 x g, Room temperature, 00:30:00 .
30m
After centrifugation, a brown pellet should form on the bottom of the tube. Decant the supernatant and invert the tube on a paper towel for 00:05:00 .
5m
Anion exchange clean-up
35m
Add 1 mL column equilibration buffer to the pellet and incubate the sample at 45 °C for 00:10:00 . Afterwards, dissolve the pellet by vortexing and/or pipetting.
10m
Place a HiPure or JetStar 2.0 column in a 15 mL centrifuge tube or a suitable rack.
Note
Racks can be bought at ThermoFisher or Qiagen but are really expensive considering it is only a piece of plastic. We plan on building our own rack for this protocol and publishing a 3D printable model.
Note
The columns seem to be identical. Unfortunately, they are not sold separately but the Kit with the HiPure mini columns is way more affordable than the original kit from Qiagen (approx. 120€ for 25 preps). We are working on a solution to clean and reuse these columns further reducing the costs of this protocol.
Equilibrate the column with 2 mL column equlibration buffer . Wait until all of the buffer volume has passed the column by gravity flow.
5m
Load the sample from step 15 on the equilibrated column. Make sure all that the pellet is dissolved properly or it might heavily reduce the flow-through and reduce the expected yield. Wait until all of the buffer volume has passed the column by gravity flow.
5m
Wash the column with 1 mL column equilibration buffer . Wait until all of the buffer volume has passed the column by gravity flow.
5m
Transfer the column to a new 15 mL centrifuge tube or place a 2 mL collection tube in the rack to collect the RNA.
Add 1 mL RNA elution buffer to the column. Wait until all of the buffer volume has passed the column by gravity flow.
5m
Transfer the column to a new 15 mL centrifuge tube or place a new 2 mL collection tube in the rack to collect the DNA
Add 1 mL DNA elution buffer to the column. Wait until all of the buffer volume has passed the column by gravity flow.
5m
Nucleic acid precipitation
50m
If a 15 mL centrifuge tube was used for elution, transfer the DNA/RNA sample to a 2 mL tube.
5m
Add 1 mL of isopropanol and incubate at -20 °C for 00:15:00
15m
13.000 x g, Room temperature, 00:15:00
15m
Decant the supernatant and invert the tubes on a paper towel for 00:10:00 to dry the DNA/RNA pellet. The RNA pellet should be barely visible and white to transparent, while the DNA pellet is mostly brownish.
10m
Resuspend the DNA/RNA in 100 µL elution buffer . Check the integrity of the DNA/RNA on an agarose gel. afterward
Note
There is usually a low amount of DNA in the RNA sample, while there is always some RNA carry-over to the DNA sample. If RNA-free DNA is needed consider digesting it with RNase A and performing a bead-cleanup afterwards (e.g. Bead-cleanup protocol for DNA). We will also publish a similar protocol for the cleanup of RNA samples.
Expected result
In the gel picture, 2 clear RNA bands and an HMW DNA band should be visible. For 5 g of sediment sample, the protocol typically yields ~ 5 µg of DNA and RNA.
Exemplary gel picture after the extraction. 4 replicate samples were extracted to compare the JetStar and HiPure columns. Residual RNA can be seen in the DNA samples.
The same samples after RNase A digestion and bead-cleanup for DNA and RNA samples. Residual RNA in the DNA is gone, RNA bands look more distinct now.
5m