TITLE: RNA/DNA extraction from plankton natural samples using NucleoSpin RNA + RNA/DNA Buffer kits (Macherey Nagel)
AUTHORS: Sarah Romac, Morgane Ratin

[DESCRIPTION]
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.


[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.


[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) ;

  

- NucleoSpin® RNA/DNA Buffer Set (Macherey-Nagel, ref 740944) (100 preps).

  



[STEPS]
SECTION: Lysis
1. Lyse your plankton filter samples following the protocol :

https://www.protocols.io/edit/cryogrinding-protocol-mecanic-lysis-of-142mm-filte-beqpjdvn


SECTION: Lysis
2. 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) ;


SECTION: Lysis
3. 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.
5 min Room temperature 

SECTION: Lysis
3. In the Falcon50 tube containing the filter powder, add 500µL of RA1 and 5µL of β-mercaptoethanol per sample. Vortex thoroughly.

Incubate at room temperature for about 5 minutes.
5 min Room temperature 

SECTION: Lysis
4. Transfer the lysate and the filter on a Nucleospin Filter (violet ring) (from  NucleoSpin RNA Mini kit) placed in a Collection Tube 2mL.

Centrifuge 1 min at 11000g at 19°C. 
11000 x g, 1 min, 19 °C 

SECTION: Lysis
5. Discard the Nucleospin Filter.

Transfer the eluate in a new sterile microtube 2mL. 

Volume is around 550 µL .
SECTION: Precipitation / Purification
6. Add 700µL EtOH 70% to the filtrate  and pipette up and down at least 5 times to mix.

Volume is around   1250 µL .
SECTION: Precipitation / Purification
7. Transfer 600µL from the lysate/ethanol-mix in a NucleoSpin RNA column (light blue ring) placed in a Collection Tube. (from  NucleoSpin RNA Mini kit)

Centrifuge 30s at 11000g at 19°C.
11000 x g, 30 s, 19 °C 

Discard flow through and replace the NucleoSpin RNA column on the Collection Tube.
SECTION: Precipitation / Purification
8. Repeat Step 7 until all the volume lysate/ethanol-mix have been passed through the NucleoSpin RNA column (light blue ring).
SECTION: DNA Purification and Elution
9. Add 500µL DNA Wash to the NucleoSpin RNA column.

Centrifuge 1min at 11000g.
11000 x g, 1 min, 19 °C 

Discard the flow-through and reuse Collection Tube;
SECTION: DNA Purification and Elution
10. Add again 500µL DNA Wash to the NucleoSpin RNA column.

Centrifuge 1min at 11000g. 
11000 x g, 1 min, 19 °C 

Discard the flow-through and place the Column in a 1.5mL sterile microtube correctly annotated (Sample code, DNA extraction date, operator).
SECTION: DNA Purification and Elution
11. Let dry the column for 3min at room temperature.
3 minRoom temperature 

SECTION: DNA Purification and Elution
12. Add 100µL DNA Elute (preheated to 50°C) directly onto the membrane and incubate 1 min at room temperature.
1 min  Room temperature 

Centrifuge 1min at 11000g;
11000 x g, 1 min, 19 °C 

Place the NucleoSpin RNA MINI column into ta new 15mL Collection tube for the following DNA digestion (see Step 14).


SECTION: DNA Purification and Elution
13. Store DNA extracts at -20°C.
100 µL 
-20 °C 
SECTION: DNA Digestion
14. Prepare DNase mix: 90µL Reaction Buffer for DNase + 10µL reconstituted rDNase per sample and mix gently.
 - for 10 samples: 900µl + 10µl ;
 - for 17 samples: 1530µl + 170µl ;


    Apply 95µL DNase Mix directly onto the center of the column and incubate 15 min at room temperature.
    15 min Room temperature 
SECTION: DNAase Inactivation and RNA purification
15. Add 200µL RAW2 (inactivation of the rDNase). 
Incubate at room temperature for 1min.

 Centrifuge 30s at 11000g.
11000 x g, 30 s, 19 °C

Place the Column into a new Collection Tube. 
SECTION: DNAase Inactivation and RNA purification
16. Add 600µL RA3.

Centrifuge 30s at 11000g.
11000 x g, 30 s, 19 °C 

Discard flow-through and replace the column into the Collection Tube.
SECTION: DNAase Inactivation and RNA purification
17. Add 250µL RA3.

Centrifuge 2min at 11000g.
11000 x g, 2 min, 19 °C 

Discard flow-through and place the Column in a sterile microtube correctly annotated (Sample code, DNA extraction date, operator).
SECTION: RNA Elution
18. Add 30µL RNase-free water (preheated to 50°C) directly onto the membrane .

Incubate 1min at room temperature.
1 min Room temperature 

 Centrifuge 1min at 11000g.
11000 x g, 1 min, 19 °C 

SECTION: RNA Elution
19. Repeat step 18 by eluting with the same eluate.

Discard the NucleoSpin RNA MINI column.
SECTION: RNA Elution
20. Store RNA extracts at -80°C.
SECTION: Lysis
4. 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, 10 min, 19 °C 

SECTION: Lysis
5. Discard the Nucleospin Filter.

Transfer the eluate in a new sterile Falcon15mL tube. 

Volume is around 3.7 mL .
SECTION: Precipitation / Purification
6. Add 4 mL EtOH 70% to the filtrate  and and vortex 2 x 5 sec to mix.

Volume is around   7.7 mL .
SECTION: Precipitation / Purification
7. 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, 3 min, 19 °C 

Discard flow through and replace the NucleoSpin RNA MIDI column on the Collection Tube.
SECTION: Precipitation / Purification
8. Repeat Step 7 until all the volume lysate/ethanol-mix have been passed through the NucleoSpin RNAMDI column.
SECTION: DNA Purification and Elution
9. Add 2.5mL DNA Wash to the NucleoSpin RNA MDI column.

Centrifuge 3min at 4500g at 19°C.
4500 x g, 3 min, 19 °C 

Discard the flow-through and reuse Collection Tube;
SECTION: DNA Purification and Elution
10. Add again 2.5mL DNA Wash to the NucleoSpin RNA column.

Centrifuge 3min at 4500g at 19°C.
4500 x g, 3 min, 19 °C 

Discard the flow-through and place the Column in a new sterile Falcon15mL.
SECTION: DNA Purification and Elution
11. Let dry the column for 5min at room temperature.
5 minRoom temperature 

SECTION: DNA Purification and Elution
12. Add 300µL DNA Elute (preheated to 50°C) directly onto the membrane and incubate 5 min at room temperature.
5 min  Room temperature 

Centrifuge 3min at 11000g;
11000 x g, 3 min, 19 °C 

Place the NucleoSpin RNA MIDI column into ta new 15mL Collection tube for the following DNA digestion (see Step 14).
SECTION: DNA Purification and Elution
13. 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.
SECTION: DNA Digestion
14. 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.
    15 min Room temperature 
SECTION: DNAase Inactivation and RNA purificationn
15. Add 2.6mL RAW2 (inactivation of the rDNase). 
Incubate at room temperature for 1min.

 Centrifuge 3min at 4500g.
4500 x g, 3 min, 19 °C

Place the Column into a new Collection Tube. 
SECTION: DNAase Inactivation and RNA purificationn
16. Centrifuge 3minmin at 454500g.
4500 x g, 3 min, 19 °C

PlaPce the ColCmn into a na newCollection Tube.  
SECTION: DNAase Inactivation and RNA purificationn
17. Add 2.6mL RA3.

Centrifuge 5min at 4500g.
4500 x g, 5 min, 19 °C


Discard flow-through and place the NucleoSpin RNA MIDI column into ta new  Falcon 15mL tube.
SECTION: RNA Elution
18. Add 300µL RNase-free water (preheated to 50°C) directly onto the membrane .

Incubate 2min at room temperature.
2 min Room temperature 

 Centrifuge 3min at 4500g.
4500 x g, 3 min, 19 °C 

SECTION: RNA Elution
19. Repeat step 18 by eluting with the same eluate.

Discard the NucleoSpin RNA MIDI column.
SECTION: RNA Elution
20. 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.
SECTION: Lysis
3. 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.
5 min Room temperature 

SECTION: Lysis
4. 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, 10 min, 19 °C 

SECTION: Lysis
5. Discard the Nucleospin Filter.

Transfer the eluate in a new sterile Falcon15mL tube. 

Volume is around 3.7 mL .
SECTION: Precipitation / Purification
6. Add 4 mL EtOH 70% to the filtrate  and and vortex 2 x 5 sec to mix.

Volume is around   7.7 mL .
SECTION: Precipitation / Purification
7. Transfer 600µL from the lysate/ethanol-mix in a NucleoSpin RNA column (light blue ring) placed in a Collection Tube. (from  NucleoSpin RNA Mini kit)

Centrifuge 30s at 11000g at 19°C.
11000 x g, 30 s, 19 °C 

Discard flow through and replace the NucleoSpin RNA column on the Collection Tube.
SECTION: Precipitation / Purification
8. Repeat Step 7 until all the volume lysate/ethanol-mix have been passed through the NucleoSpin RNA column (light blue ring).
SECTION: DNA Purification and Elution
9. Add 600µL DNA Wash to the NucleoSpin RNA column.

Centrifuge 1min at 11000g.
11000 x g, 1 min, 19 °C 

Discard the flow-through and reuse Collection Tube;
SECTION: DNA Purification and Elution
10. Add again 600µL DNA Wash to the NucleoSpin RNA column.

Centrifuge 1min at 11000g. 
11000 x g, 1 min, 19 °C 
SECTION: DNA Purification and Elution
12. Let dry the column for 3min at room temperature.
3 minRoom temperature 

SECTION: DNA Purification and Elution
13. Add 100µL DNA Elute (preheated to 50°C) directly onto the membrane and incubate 2 min at room temperature.
2 min  Room temperature 

Centrifuge 1min at 11000g;
11000 x g, 1 min, 19 °C 

Place the NucleoSpin RNA MINI column into ta new 2mL Collection tube for the following DNA digestion (see Step 15).


SECTION: DNA Purification and Elution
14. Take an aliquot of 5µL for quantification (Qubit, Bioanalyzer, and Nanodrop).
Store DNA extracts at -20°C.
100 µL 
-20 °C 
SECTION: DNA digestion
15. Prepare DNase mix: 90µL Reaction Buffer for DNase + 10µL reconstituted rDNase per sample and mix gently.
 - for 10 samples: 900µl + 10µl ;
 - for 17 samples: 1530µl + 170µl ;


    Apply 95µL DNase Mix directly onto the center of the column and incubate 15 min at room temperature.
    15 min Room temperature 
SECTION: DNAase Inactivation and RNA purification
16. Add 200µL RAW2 (inactivation of the rDNase). 
Incubate at room temperature for 1min.

 Centrifuge 30s at 11000g.
11000 x g, 30 s, 19 °C

Place the Column into a new Collection Tube. 
SECTION: DNAase Inactivation and RNA purification
17. Add 600µL RA3.

Centrifuge 30s at 11000g.
11000 x g, 30 s, 19 °C 

Discard flow-through and replace the column into the Collection Tube.
SECTION: DNAase Inactivation and RNA purification
19. Add 600µL ETOH 96-100%.

Centrifuge 30s at 11000g.
11000 x g, 30 s, 19 °C 

Discard flow-through and replace the column into the Collection Tube.
SECTION: RNA elution
21. Add 30µL RNase-free water (preheated to 50°C) directly onto the membrane .

Incubate 2min at room temperature.
2 min Room temperature 

 Centrifuge 1min at 11000g.
11000 x g, 1 min, 19 °C 

SECTION: RNA elution
22. Repeat step 21 by eluting with the same eluate.

Discard the NucleoSpin RNA MINI column.
SECTION: RNA elution
23. Process directly to the TurboDNase treatment (Turbo-DNAfreeTM kit, Ambion AM1907).
SECTION: DNA Purification and Elution
11. Add again 600µL DNA Wash to the NucleoSpin RNA column.

Centrifuge 1min at 11000g. 
11000 x g, 1 min, 19 °C 

Discard the flow-through and place the Column in a 1.5mL sterile microtube correctly annotated (Sample code, DNA extraction date, operator).
SECTION: DNAase Inactivation and RNA purification
18. Add 600µL RA3.

Centrifuge 30s at 11000g.
11000 x g, 30 s, 19 °C 

Discard flow-through and replace the column into the Collection Tube.
SECTION: DNAase Inactivation and RNA purification
20. Centrifuge 2min at 11000g.
11000 x g, 2 min, 19 °C 

Discard flow-through and place the Column in a 1.5mL sterile microtube correctly annotated (Sample code, RNA extraction date, operator).