Dec 07, 2023
Version 2
RNA/DNA extraction from plankton natural samples using NucleoSpin RNA + RNA/DNA Buffer kits (Macherey Nagel) V.2
- 1UMR7144-Roscoff Marine Station;
- 2Station Biologique, CNRS, Roscoff
- Morgane Ratin: Morgane contributes to the optimization of the "Step-case [<20µm]-142mm filters";
Protocol Citation: Sarah Romac, Morgane Ratin 2023. RNA/DNA extraction from plankton natural samples using NucleoSpin RNA + RNA/DNA Buffer kits (Macherey Nagel). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynz2qvx9/v2Version created by Sarah Romac
Manuscript citation:
Alberti A., Poulain J., Engelen S., Labadie K., Sarah Romac S., [...], Wincker P. Viral to metazoan marine plankton nucleotide sequences from the TaraOceans expedition. Scientific Data (2017). dx.doi.org/10.17504/protocols.io.qv6dw9e
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: December 06, 2023
Last Modified: December 07, 2023
Protocol Integer ID: 91882
Abstract
This protocol has been developped for simultaneous nucleic acid (RNA and DNA) extraction from environmental water samples collected by filtration on polycarbonate 47mm and 142mm filters.
It has been developped during BioMarks project (2009-2013) and has been used in many projects studying plankton diversity (TARA-OCEANS 2009-2013, TARA-PACIFIC 2016-2018, TONGA 2019), and plankton monitoring projects (MOOSE-GE 2017- ongoing).
This protocol is applied in routine in our lab and also in CEA Genoscope.
Guidelines
This protocol has been modified from 3 references provided by Macherey-Nagel :
- NucleoSpin RNA Mini kit (Macherey-Nagel, ref 740955.50) (50 preps) ;
- NucleoSpin RNA Midi kit (Macherey-Nagel, ref 740962.20) (20 preps) ;
Instruction-NucleoSpin-RNA.pdf - NucleoSpin® RNA/DNA Buffer Set (Macherey-Nagel, ref 740944) (100 preps).
Instruction-NucleoSpin-RNA-DNA-Buffer-Set.pdf Materials
Kits and reagents :
- NucleoSpin RNA Mini kit (Macherey-Nagel, ref 740955.50) (50 preps) :
-Store lyophilized RDNase A at +4°C on arrival;
-Store all other kits components at room-temperature.
- NucleoSpin RNA Midi kit (Macherey-Nagel, ref 740962.20) (20 preps) :
-Store lyophilized RDNase A at +4°C on arrival;
-Store all other kits components at room-temperature.
- NucleoSpin® RNA/DNA Buffer Set (Macherey-Nagel, ref 740944) (100 preps).
- β-mercaptoethanol (Sigma-Aldrich, ref 63689-25ML-F).
Specific equipments :
- Chemical hood Captair MID CAP 633 (Erlab)
- Centrifuge 5417R equipped with rotor 30 wells 1.5-2mL microtubes (Eppendorf)
- Centrifuge 5804R equipped with Swing Rotor 16 wells Falcon 15mL tubes (Eppendorf)
- Thermomixer equipped with Thermoblock 24 tubes 1.5mL (Eppendorf)
Safety warnings
Always wear a labcoat, and clean gloves for each step.
Decontaminate all the area (chemical hood, centrifuges, thermomixer, pipettes, and racks) with RNase away before to start.
Always use filter tips and sterile 1.5mL microtubes.
Work under a chemical hood.
Before start
- rDNase: add the volume of RNase-free H2O recommended from each kit, incubate 1min at Room Temperature.
Don't vortex!
Do aliquots of 150µL and store at -20°C. Do not freeze/thaw the aliquots more than 3 times.
- Wash Buffer RA3: add 50ml EtOH 96-100%.
- DNA Wash Buffer: add 90ml EtOH 50%.
- Cool centrifuge to 19°C
- Warm up DNA Elute Buffer and Nuclease-free water to 50°C.
- Check if clean cisors (DNA/RNA free) are available.
- Heat the Thermomixer (Eppendorf) to 50°C for DNA elution.
Lysis
Lysis
6m
Lyse your plankton filter samples following the protocol :
Depending of the plankton size fraction samples you have, select :
1 - RNA/DNA extraction using NucleoSpin RNA MIDI kit regarding [>20µ] size fractions (samples collected and prefiltered using a plankton net) ;
2 - RNA/DNA extraction using NucleoSpin RNA MIDI and MINI kits regarding [<20µ] size fractions (samples collected using NIskin bottles or pumping and filtered on 142mm filters) ;
3 - RNA/DNA extraction using NucleoSpin RNA MINI kits regarding [<20µ] size fractions (samples collected using NIskin bottles or pumping and filtered on 47mm filters) ;
1 _ [>20] - 47mm - NucleoSpin RNA MIDI18 steps
1 - RNA/DNA extraction using NucleoSpin RNA MIDI kit regarding [>20µ] size fractions (samples collected and prefiltered using a plankton net).
In the Falcon50 tube containing the filter powder, add 3.6 mL of RA1 and 36 µL of β-mercaptoethanol per sample. Vortex thoroughly.
Incubate at room temperature for about 5 minutes.
00:05:00 Room temperature
5m
Transfer the lysate and the filter on a Nucleospin Filter placed in a Collection Tube 15 mL. (from NucleoSpin RNA MIDI kit)
Centrifuge 10 min at 4500g at 19°C
4500 x g, 19°C, 00:10:00
10m
Discard the Nucleospin Filter.
Transfer the eluate in a new sterile Falcon15mL tube.
Volume is around 3.7 mL .
Precipitation / Purification
Precipitation / Purification
6m
Add 4 mL EtOH 70% to the filtrate and and vortex 2 x 5 sec to mix.
Volume is around 7.7 mL .
Transfer 3 mL from the lysate/ethanol-mix in a NucleoSpin RNA MIDI column placed in a Collection Tube 15mL. (from NucleoSpin RNA MIDI kit)
Centrifuge 3min at 4500g at 19°C.
4500 x g, 19°C, 00:03:00
Discard flow through and replace the NucleoSpin RNA MIDI column on the Collection Tube.
3m
Repeat Step 7 until all the volume lysate/ethanol-mix have been passed through the NucleoSpin RNAMDI column.
DNA Purification and Elution
DNA Purification and Elution
6m
Add 2.5mL DNA Wash to the NucleoSpin RNA MDI column.
Centrifuge 3min at 4500g at 19°C.
4500 x g, 19°C, 00:03:00
Discard the flow-through and reuse Collection Tube;
3m
Add again 2.5mL DNA Wash to the NucleoSpin RNA column.
Centrifuge 3min at 4500g at 19°C.
4500 x g, 19°C, 00:03:00
Discard the flow-through and place the Column in a new sterile Falcon15mL.
3m
Let dry the column for 5min at room temperature.
00:05:00 Room temperature
5m
Add 300µL DNA Elute (preheated to 50°C) directly onto the membrane and incubate 5 min at room temperature.
00:05:00 Room temperature
Centrifuge 3min at 11000g;
11000 x g, 19°C, 00:03:00
Place the NucleoSpin RNA MIDI column into ta new 15mL Collection tube for the following DNA digestion (see Step 14).
8m
Transfer the DNA extract on a sterile 1.5mL sterile microtube correctly annotated (Sample code, DNA extraction date, operator).
Store DNA extracts at -20°C.
DNA Digestion
DNA Digestion
6m
Prepare DNase mix: 235µL Reaction Buffer for DNase + 25µL reconstituted rDNase per sample and mix gently.
- for 10 samples: 2350µl + 250µl ;
- for 17 samples: 3995µl + 425µl ;
Apply 235µL DNase Mix directly onto the center of the column and incubate 15 min at room temperature.
00:15:00 Room temperature
DNAase Inactivation and RNA purificationn
DNAase Inactivation and RNA purificationn
6m
Add 2.6mL RAW2 (inactivation of the rDNase).
Incubate at room temperature for 1min.
Centrifuge 3min at 4500g.
4500 x g, 19°C, 00:03:00
Place the Column into a new Collection Tube.
3m
Centrifuge 3minmin at 454500g.
4500 x g, 19°C, 00:03:00
PlaPce the ColCmn into a na newCollection Tube.
3m
Add 2.6mL RA3.
Centrifuge 5min at 4500g.
4500 x g, 19°C, 00:05:00
Discard flow-through and place the NucleoSpin RNA MIDI column into ta new Falcon 15mL tube.
5m
RNA Elution
RNA Elution
6m
Add 300µL RNase-free water (preheated to 50°C) directly onto the membrane .
Incubate 2min at room temperature.
00:02:00 Room temperature
Centrifuge 3min at 4500g.
4500 x g, 19°C, 00:03:00
5m
Repeat step 18 by eluting with the same eluate.
Discard the NucleoSpin RNA MIDI column.
Transfer the RNA extract on a sterile 1.5mL sterile microtube correctly annotated (Sample code, RNA extraction date, operator).
Store RNA extract at -80°C.
Protocol references
Alberti, A., Poulain, J., Engelen, S., Labadie, K., Romac, S., Ferrera, I., Albini, G., Aury, J. M., Belser, C., Bertrand, A., Cruaud, C., Da Silva, C., Azema-Dossat, C., Gavory, F., Gas, S., Guy, J., Haquelle, M., Jacoby, E., Jaillon, O., Lemainque, A., Pelletier, E., Samson, G., Wessner, M., Acinas, S., Royo-Llonch, M., Cornejo-Castillo, F., Logares, R., Fernandez-Gomez, B., Bowler, C., Cochrane, G., Amid, C., Hoopen, P. Ten, de Vargas, C., Grimsley, N., Desgranges, E., Kandels-Lewis, S., Ogata, H., Poulton, N., Sieracki, M., Stepanauskas, R., Sullivan, M., Brum, J., Duhaime, M., Poulos, B., Hurwitz, B., Pesant, S., Karsenti, E. & Wincker, P. Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. Sci. Data, 4, 170093 (2017).
Câmara dos Reis M. et al. 2022. Exploring the phycosphere of Emiliania huxleyi: from
bloom dynamics to microbiome assembly experiments. DOI: 10.1101/2022.02.21.481256