TITLE: eRNA extraction from water samples filtered through 47 mm diameter filters  (NucleoMag DNA/RNA Water Kit - MACHEREY NAGEL).
AUTHORS: Marine Vautier, Cecile Chardon, Cyrielle Galiegue, Isabelle Domaizon

[DESCRIPTION]
The objective of this protocol is the environmental RNA (eRNA) extraction from water samples filtered through 47 mm diameter filters. 

RNA extraction is performed using a MagnetaPure 32 Nucleic Acid Purification System (Dutscher) and with the NucleoMagDNA/RNA Water Kit (Macherey Nagel), with a rDNase digestion step.

The procedure is based on reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions. 

The benefits of using the MagnetaPure 32 Nucleic Acid Purification System (Dutscher) are increased productivity and repeatability, as well as eliminating human error and the pain of repetitive work.

This protocol is used prior to molecular biology analysis (e.g. qPCR, metabarcoding, ddPCR) to specifically target both macro- and micro-organisms eRNA extracted from water samples.


[BEFORE_START]
Filtration and preservation of the water samples through filters

As eRNA degrades very rapidly, the water samples must be filtered and preserved very quickly after sampling (less than one hour if possible). If filtration is carried out in the field, the filters should be immediately frozen in liquid nitrogen and then stored at -80°C until the eRNA is extracted. If filtration is carried out in the laboratory, the filters can be stored at -80°C immediately after filtration and until the eRNA is extracted.

Note: As eRNA is very sensitive to RNAse, which is active at temperatures above -70°C, it is very important to freeze the filters in liquid nitrogen or at -80°C very quickly after filtration.

This protocole is suitable for the RNA extraction from different types of 47 mm diameter filters:  
 

The filters should be preserved in 5 mL tubes for this RNA extraction protocol.

The following precautions must be applied:

- Wear gloves throughout the extraction process
- Clean the bench with a RNA-removing solution (e.g. RNase-off).
- Use tips with filters to avoid contaminations
- All steps have to be performed under a specific DNA/RNA-work station (sterile area equipped with air filtration and UV systems)

For any manipulation in a rare DNA/RNA room, provide complete equipment (disposable coat, cap, mask, shoe covers & gloves).

Pre and post extraction equipment decontamination:

      - Clean a specific DNA/RNA-work station and apply UV
      - MagnetaPure 32 System (Dutscher): Visual check for residues to be removed and UV decontamination – see instrument manual 

Scissors decontamination (to be done before starting the protocol, and between each filter cutting)

- Prepare : 
one 50 mL tube with RNA-removing solution (e.g. DNA/RNase-off, DNA/RNA away)
one 50 mL tube with molecular biology grade water
one 50 mL tube with ethanol
- Successively dip the scissors into each tube, opening and closing the scissors in each tube.

Note: The cutting of filters is not a requirement, but it does have an impact on the extraction efficiency.



[GUIDELINES]
The main steps of the protocol are:

Material and rDNase preparation
Plate preparation
Sample lysis
eRNA extraction with the MagnetaPure 32 System (Dutscher)
eRNA elution

[STEPS]
SECTION: Material and rDNase preparation
1. Material  preparation

To limit contamination of the kit buffers, it is recommended to aliquote them:
- Into 50 mL tubes for MWA1, MWA2, MWA3 and MWA4
- Into 2 mL tubes for NucleoMag B-Beads solution and RNase-free H2O

Tubes annotation 
- one 2 mL tube per sample for lysate collection
- one 5 mL tube per 8 samples for the NucleoMag B-Beads and MWA2 mix preparation
- three 0.5 mL tubes per sample for RNA collection

Scissors decontamination (to be done before starting the protocole, and between each filter cutting)
- Prepare : 
one 50 mL tube with RNA-removing solution (e.g. RNase-off)
one 50 mL tube with molecular biology grade water
one 50 mL tube with ethanol
- Successively dip the scissors into each tube, opening and closing the scissors in each tube.

Note: The cutting of filters is not a requirement, but it does have an impact on the extraction efficiency.

SECTION: Plate preparation 1/2
3. 
In this step, the buffers provided by the kit are distributed in a 96-well plate.
For RNA extraction from filtered water samples, the 12 columns of the plate are divided into 2 sections of 6 columns each, allowing up to 16 samples to be extracted per plate.

Annotate the 96-well plate as recommended below:

 

Note: It is useful to mark the dividing line between columns 6 and 7 with a marker pen to provide a visual cue for filling the plate.

Add the appropriate buffers into the appropriate wells of the plate (Table 2):

1stcolumn / 7thcolumn: Will be filled during plate preparation 2/2
2ndcolumn / 8thcolumn:   850 µL of MWA3 (1st wash)
3rdcolumn / 9thcolumn:   850 µL of MWA3 (2nd wash)
4thcolumn /10thcolumn: Will be filled during plate preparation 2/2
5thcolumn / 11thcolumn:  850 µL of MWA4 (3rd wash and bead drying)
6thcolumn / 12thcolumn: 50 µL or 65 µL of RNase-free  H2O (RNA elution)

Note: The choice of elution volume is based on the expected eRNA amount. The smaller the amount, the smaller the elution volume in order to obtain more concentrated RNA.


 

Film and reserve the plate at Room temperature
SECTION: Sample Lysis
5. During this step, a mechanical and chemical lysis of the sample is performed.


- Collect the 5mL tubes containing the filters from the freezer and place them on ice 
- Add immediately 750 µL of MWA1 buffer into each tube
- Cut the filter into small pieces directly into each tube using decontaminated scissors
Note: Scissors must be decontaminated between each sample (see section 1 "material preparation")
- Place the tubes on the vertical vortex 5 s at median speed
- Place the tubes on the horizontal vortex 5 min at maximum speed
- Place the 5 mL tubes into the benchtop centrifuge
- Pipette the lysate from the tube (approximately 650 µL) and transfer it into a 2 mL tube
- Centrifuge at   
- Replace the tubes into the rack and reserve them at Room temperature until their distribution into the 96-well plate

SECTION: Plate preparation 2/2
6. Preparation of the NucleoMag B-Beads and MWA2 mix 
NucleoMag B-Beads and MWA2 mix sediment quickly, vortex between each samples to ensure homogeneity     

- Vigorously vortex NucleoMag B-Beads tube 
- For each tube containing MWA2 buffer (previously prepared), add the appropriate volume of NucleoMag B-Beads (see Table 4 above)  
- Vortex

NucleoMag B-Beads - MWA2 mix and lysate distribution
- Remove the film from the plate
- Add the appropriate solution into each well of the plate 

4thcolumn /10thcolumn:
500 µL of NucleoMag B-Beads and MWA2 mix 
450 µL of Lysate (supernatant from the 2 mL tubes)

rDNase mix distribution
- Slowly agitate the rDNase mix tube (previously prepared- see step 2)
- Add the appropriate solution into each well of the plate 

1stcolumn / 7thcolumn:
300 µL of rDNase  mix

 

SECTION: eRNA extraction with the MagnetaPure 32 System (Dutscher)
7. Extraction part 1
- Place the plate into the MagnetaPure 32 System and insert the magnetic rod coverfor – see instrument manual

- Select the appropriate program for RNA extraction part 1
 
- Start the run (The run lasts approximately 35 min)

At the end of DNA digestion
- Remove the plate and add the appropriate solution into each well of the plate 

1stcolumn / 7thcolumn: 350 µL of MWA2
 

Extraction part 2
- Place the plate back into the MagnetaPure 32 System (it is not necessary to change the magnetic rod coverfor)

- Select the appropriate program for RNA extraction part 2

 

- Start the run (The run lasts approximately 30 min)

SECTION: Transfer of eRNA extracts
8. - At the end of the run, remove the plate and place it on ice into the DNA/RNA-workstation
- Remove the magnetic rod cover and start UV for decontamination – see instrument manual
- In the DNA/RNA-workstation, transfer each eRNA extract into a 0.5 mL tube previously annotated.

Note: It is recommended to aliquot the eRNA extracts directly to avoid freezing and thawing cycles that could degrade the eRNA.

Note:  RNA concentration and quality can be measured at this step (e.g. Nanodrop)

- Store eRNA extracts at -80 °C 

SECTION: Plate preparation 1/2
4. Preparation of the NucleoMag B-Beads and MWA2 mix:
- Prepare one 5 mL tube / maximum 8 samples  
- Add MWA2 only, the NucleoMag B-beads will be added during plate preparation 2/2 (allow a margin of one sample for the mix preparation. For example: plan a mix for 9 samples if 8 samples are to be extracted)

 

SECTION: Material and rDNase preparation
2. rDNase preparation

Reconstitution of lyophilized rDNase:
- Add 4 mL of reaction buffer for rDNase to the rDNase vial
- Incubate for 2 min - 3 min at Room temperature 
- Gently shake the vials to completely dissolve the rDNase
Note: Be careful not to mix the rDNase vigorously because the rDNase is sensitive to mechanical agitation

2. rDNAse Dilution:
- Transfer the reconstituted rDNase to a 50 mL  tube
- Add 28 mL  of reaction buffer for rDNase
- Gently shake the tube

Note: The resulting rDNase solution will be sufficient for 96 samples. If fewer than 96 samples are to be processed, it is advisable to aliquot the rDNase as it should not be frozen and thawed more than three times. Alliquots can be store at -20 °C for at least 6 months.