Sep 19, 2023

Public workspaceCo-extraction of RNA and DNA from soil and sediment samples V.2

DOI
dx.doi.org/10.17504/protocols.io.rm7vzb6m4vx1/v2
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research;
  • 2University of Duisburg-Essen
Dominik Buchner
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzb6m4vx1/v2
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
Abstract
This protocol describes extracting RNA and DNA from soil and sediment samples. Amount2 g of soil or up to Amount5 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
Download Column_Rack_PureLink_HiPure_Druck.stlColumn_Rack_PureLink_HiPure_Druck.stl377KB
Download Column Rack PureLink HiPure Bottom.stlColumn Rack PureLink HiPure Bottom.stl1KB

Chemicals:
Sodium phosphate dibasic ReagentSodium phosphate dibasicSigma AldrichCatalog #S0876-100G
Guanidinium thiocyanate ReagentGuanidinium thiocyanateFisher ScientificCatalog #10503345
Sodium phosphate monobasic ReagentSodium phosphate monobasicSigma AldrichCatalog #S0751-100G
SDS ultrapure ReagentSodium dodecyl sulfateDiagonalCatalog #A1112.0500
Sodium chloride ReagentSodium chlorideFisher ScientificCatalog #10616082
Tris ultrapure 99.9% ReagentTris ultrapure 99.9%DiagonalCatalog #A1086.1000
Hydrochloric acid fuming 37% ReagentHydrochloric acid fuming 37%Sigma AldrichCatalog #1003171011
Aluminium ammonium sulfate dodecahydrate alReagentAluminium ammonium sulfate dodecahydrateSigma AldrichCatalog #A2140-500G
Phenol/chloroform/isoamyl alcohol Ph8 ReagentROTI Phenol/Chloroform/Isoamyl alcoholCarl RothCatalog #A156.3
Tri-Sodium citrate Reagenttri-Sodium citrateSigma AldrichCatalog #1110371000
Citric acid ReagentCitric acidSigma AldrichCatalog #251275-100G
MOPS ReagentMOPSCarl RothCatalog #6979.3
Sodium hydroxide ReagentSodium Hydroxide PelletsFisher ScientificCatalog #S318
PureLink HiPure Plasmid-MiniPrep-Kit ReagentPureLink HiPure Plasmid-MiniPrep-KitThermo Fisher ScientificCatalog #K210002


Labware:
15 mL centrifuge tubes, Ultra-High PerformanceReagentCentrifuge tubes Ultra-High PerformanceVWR AvantorCatalog #525-1091
0.1 mm glass beads ReagentGlass Beads 0.1 mm diaBioSpec ProductsCatalog #11079101
0.5 mm glass beads ReagentGlass Beads 0.5 mm diaBioSpec ProductsCatalog #11079105
1 mm zirconia/silica beads ReagentZirconia/Silica Beads 1 mm diaBioSpec ProductsCatalog #11079110z
2 mm zirconia beads ReagentZirconia Beads 2 mm diaBioSpec ProductsCatalog #11079124zx



Stock solutions:
Amount1 L SDS stock solution Concentration10 Mass / % volume
  • Add Amount100 g SDS ultrapure to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

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
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8 with HCl
  • 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 Amount121.14 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
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL trisodium citrate stock solution Concentration300 millimolar (mM) Ph5
  • Add Amount38.7 g tri-Sodium citrate to a beaker
  • Adjust pH to Ph5 with citric acid
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL MOPS stock solution
  • Add Amount104.64 g MOPS to a beaker
  • Adjust volume to Amount450 mL with ddH2O
  • Adjust pH to Ph7 with sodium hydroxide
  • Sterilize by filtering and store at TemperatureRoom temperature

Working solutions:
Amount500 mL bead-beating solution (Concentration180 millimolar (mM) sodium phosphate , Concentration120 millimolar (mM) guanidinium thiocyanate ) Ph8
  • Add Amount12.8 g sodium phosphate dibasic to a beaker
  • Add Amount7.1 g guanidinium thiocyanate
  • Adjust volume to Amount490 mL with ddH2O
  • Adjust pH to Ph8 by adding sodium phosphate monobasic
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL lysis solution (Concentration150 millimolar (mM) sodium chloride , Concentration4 Mass / % volume SDS , Concentration500 millimolar (mM) Tris ) Ph8
  • Add Amount200 mL of Concentration10 Mass / % volume SDS stock solution
  • Add Amount15 mL of Concentration5 Molarity (M) sodium chloride stock solution
  • Add Amount250 mL of Concentration1 Molarity (M) Tris stock solution Ph8
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL inhibitor removal solution (Concentration120 millimolar (mM) aluminium ammonium sulfate dodecahydrate )
  • Add Amount27.2 g aluminium ammonium suldate dodecahydrate to a beaker
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL precipitation solution (Concentration5 Molarity (M) sodium chloride , Concentration30 millimolar (mM) sodium citrate ) Ph5
  • Add Amount146.1 g sodium chloride to a beaker
  • Add Amount50 mL of Concentration300 millimolar (mM) tri-Sodium citrate stock solution Ph5
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL column equlibration buffer (Concentration500 millimolar (mM) sodium chloride , Concentration50 millimolar (mM) MOPS , Concentration15 % (v/v) isopropanol ) Ph7
  • Add Amount50 mL of Concentration5 Molarity (M) sodium chloride stock solution to a beaker
  • Add Amount25 mL of Concentration1 Molarity (M) MOPS stock solution Ph7
  • Add Amount75 mL isopropanol
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL RNA elution buffer (Concentration750 millimolar (mM) sodium chloride , Concentration50 millimolar (mM) MOPS , Concentration15 % (v/v) isopropanol ) Ph7
  • Add Amount75 mL of Concentration5 Molarity (M) sodium chloride stock solution to a beaker
  • Add Amount25 mL of Concentration1 Molarity (M) MOPS stock solution Ph7
  • Add Amount75 mL isopropanol
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL DNA elution buffer (Concentration1250 millimolar (mM) sodium chloride , Concentration50 millimolar (mM) Tris , Concentration15 % (v/v) isopropanol ) Ph8.5
  • Add Amount125 mL of Concentration5 Molarity (M) sodium chloride stock solution to a beaker
  • Add Amount25 mL of Concentration1 Molarity (M) Tris stock solution Ph8.5
  • Add Amount75 mL isopropanol
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L elution buffer (Concentration10 millimolar (mM) Tris ) Ph8.5
  • Add Amount10 mL Tris stock solution Ph8.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature





Safety warnings
Attention
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
Cell lysis
42m
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 Amount2.5 mL bead-beating solution , Amount0.25 mL lysis solution , Amount0.8 mL inhibitor removal solution and Amount3.5 mL phenol/chloroform/isoamyl alcohol Ph8 . 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 Duration00:15:00 .


15m
Centrifigation2500 x g, Room temperature, 00:10:00

10m
Transfer Amount3.5 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
Inhibitor removal
30m
30m
Add Amount1.5 mL precipitation solution . The mixture will be cloudy at first but will clear up after vortexing for a few seconds.



5m
Incubate at Temperature4 °C for Duration00:10:00 .

10m
Centrifigation2500 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
Nucleic acid precipitation
1h 5m
1h 5m
Add Amount5 mL isopropanol. Mix by vortexing and incubate at TemperatureRoom temperature for Duration00:30:00 .

30m
Centrifigation2500 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 Duration00:05:00 .



5m
Anion exchange clean-up
Anion exchange clean-up
35m
35m
Add Amount1 mL column equilibration buffer to the pellet and incubate the sample at Temperature45 °C for Duration00:10:00 . 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 Amount2 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 Amount1 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 Amount1 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 Amount1 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
Nucleic acid precipitation
50m
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 Temperature-20 °C for Duration00:15:00

15m
Centrifigation13.000 x g, Room temperature, 00:15:00

15m
Decant the supernatant and invert the tubes on a paper towel for Duration00:10:00 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 Amount100 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 Amount5 g of sediment sample, the protocol typically yields ~ Amount5 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