Dec 01, 2025
RNA extraction and purification from cultured diatom viruses
- Pauline Nogaret1,
- Estelle Bigeard1,
- Laure Arsenieff1,
- Anne Claire Baudoux1
- 1Station biologique de Roscoff, FRANCE, UMR7144 CNRS Sorbonne Université
- Estelle Bigeard: co-first author
- Ecology of Marine Plankton (ECOMAP) team - RoscoffTech. support email: [email protected]
Protocol Citation: Pauline Nogaret, Estelle Bigeard, Laure Arsenieff, Anne Claire Baudoux 2025. RNA extraction and purification from cultured diatom viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8k13lmk/v1
Manuscript citation:
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: June 27, 2024
Last Modified: December 01, 2025
Protocol Integer ID: 102510
Keywords: extraction, RT PCR, RNA virus, virus, chloroform purification, nucleic acid extraction, RNA viruses, marine ecology, environmental analysis, purification from cultured diatom virus, genome sequences of marine rna virus, marine rna virus, cultured diatom virus, rna extraction, infecting rna virus, rna virus, processing viral lysate, virus concentration by tangential flow filtration, virus concentration, sequencing genome, viral lysate, final nucleic acid quantification, genome sequence, based purification, purification
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-22-CE01-0018
Abstract
This protocol describes a workflow for processing viral lysates to obtain genome sequences of marine RNA viruses. The procedure includes lysate clarification, virus concentration by tangential flow filtration, chloroform-based purification, RNA extraction and conversion to cDNA, and final nucleic acid quantification. cDNA produced using this protocol is suitable for sequencing genomes of diatom-infecting RNA viruses. This protocol is adapted from the method described in Arsenieff et al. (2019).
Image Attribution
Transmission electron micrograph of M.comicus (RCC4660) infection with an Bacillarnavirus (RCC7291).
Guidelines
Materials
Equipments :
weighing machin & pH meter
Centrifuge Avanti J-25 XP & Rotor JA-10 (Bekman Coulter)
Chemical hood
Micropipets
Hand pump and pump
Eppendorf thermomixer & smartblock 1,5ml
Thermocycler
Microwave
Electrophoresis system & generator (Sub Cell Model 96 cell)
Image Quant LAZ4000 (Ge Healthcare)
Consumables:
Autoclaved 500mL Centrifuge bottles, PPCO/HDPE/PC/FEP, with screw cap, Nalgene‱ (VWR, cat no : 525-2313)
1L Ventilated Culture flask (Starlab, cat no : CC7682-4822)
0,45µm Filtration unit (VWR, cat no : 2566881-514-0343)
Vivaflow‱ 200 30Kda, MWCO, PES (Sartorius, cat no : VF20P2)
Vivaspin‱20 30Kda, MWCO, PES (Cytiva, cat no : 28932361)
Syringe filter 0,2µm 25mm, PES (Fisher scientific, cat no : 15392388)
Falcon Tubes 50mL, 15mL
Microtubes 1,5mL ,2mL, 0,2µL (Eppendorf-type)
Filter tips
Reagents:
KH2PO4 (Merck Sigma, cat no : 60218-100G)
Na2HPO4 (Merck Sigma, cat no : 71642)
Chloroform (Merck Sigma, cat no : C2432)
Isopropanol (Merck Sigma, cat no : I9516)
Absolute ethanol (Fisher scientific, cat no : 10342652)
Tris-Base (Merck Sigma, cat no : T6791)
Acetic acid (>99.7%) (Fisher Scientific, cat no : 11463473)
0,5 M EDTA pH 8,0 (Fisher Scientific, cat no : 15575-020)
Master Pure complete DNA and RNA purification kit (Epicentre, cat no : MC85200)
Qubit RNA BR Assay Kit (Invitrogen, cat no : Q10210)
Qubit dsDNA BR Assay Kit (Invitrogen, cat no : Q32853)
SuperScript III Reverse Transcriptase (Invitrogen, cat no : 18080044)
RNAse OUT Recombinant Rnase Inhibitor (Invitrogen, cat no : 10777019)
Random primers, 250ng (Invitrogen, cat no : 48190011 )
dNTP mix, 10mM (Invitrogen, cat no : 18427013 )
Primers RdRp Forward and Reverse (10µM)
DNA polymerase Taq platinium; MgCl2 (50mM); Rxn PCR buffer (10X); dNTP mix (10mM) (Fisher scientific, cat no : 10966018)
Nuclease-Free Water (Invitrogen, cat no : AM9937)
Ice
Loading buffer
Agarose (Interchim, cat no : 31272L)
Ethidium bromide (Merck Sigma, cat no : E1510)
SmartLadder - 200 to 10000 bp (Eurogentec, cat no : MW-1700-10)
Troubleshooting
Before start
Solution preparation:
cold isopropanol at -20°C
cold 70% ethanol at -20°C
10mM phosphate solution (10 mM KH₂PO₄ + 10 mM Na₂HPO₄), pH 7.2 filtered through a 0,2µm filter
TAE Buffer (Tris Acetate EDTA) 50X :
Tris-Base (2M final) : 242.5 g (Merck Sigma, cat no : T6791)
Acetic acid (>99.7%, 2M final) : 7 ml (Fisher Scientific, cat no : 11463473)
0,5 M EDTA pH 8,0 (50 mM final) : 100 ml (Fisher Scientific, cat no : 15575-020)
dH20 qsp 1L
RNA virus culture.
3w
One liter of diatom host culture is grown exponentially in K+Si medium (Keller et al., 1987) at 18 °C under a 12:12 h light:dark cycle with an irradiance of 100 µmol photons·m⁻²·s⁻¹, provided by white fluorescent light (Philips Master TL-D 18W/865). A freshly produced viral lysate, filtered through a 0.45 µm PES membrane, is added to the host culture at a final concentration of 10–20% (v/v). The infected culture is incubated under the same conditions as the host culture until complete lysis is observed.
1 L
RNA virus concentration.
3d 7h 38m
Lysate clarification :
- Centrifuge the viral lysate (2x500mL) 5353 x g, 4°C, 00:30:00 (Avanti J-25XP, Beckman Coulter-JA-10 Rotor).
- Carefully transfer the supernatant into a 0.45 µm filtration unit (Stericup), avoiding disturbance of the pellet.
- Filter the supernatant completely using a hand pump.
30m
Viral concentration :
- Concentrate the clarified viral lysate to 50 mL using a Vivaflow‱ 200 cartridge (30 kDa MWCO, PES, Sartorius).
- Further concentrate the sample to ~1 mL using a Vivaspin‱ 20 (30 kDa MWCO, PES, Cytiva) by centrifugation6000 x g, 4°C, 00:10:00
- Keep the Vivaspin at 4°C overnight.
- Rinse the Vivaspin‱ 20 by adding 1 mL of 10 mM phosphate solution, and transfer the rinse to the Falcon tube containing the sample.
- Repeat the rinsing step until a final volume of 10 mL is reached.
1d 4h
Viral Purification :
Under a chemical hood :
- Add 1 volume of chloroform (v/v, 10mL) into the Falcon tube containing the sample.
- Vortex thoroughly.
- Centrifuge. 2200 x g, 12°C
- Carefully transfer the aqueous phase (upper phase containing viruses ) into a new Falcon tube.
- Add again 1 volume of chloroform
- Repeat previous steps a total of 7 times. 03:00:00
- Leave the tube (containing 5mL of sample) open overnight under the chemical hood to evaporate residual chloroform.Overnight
- Concentrate the sample in 1mL by using Vivaspin‱206000 x g, 12°C, 00:05:00
- Collect sample and add 1mL of nuclease-free water. Centrifuge in the same Vivaspin‱206000 x g, 12°C, 00:03:00 . Repeat this whashing step 3 times.
- Incubate the Vivaspin‱20 overnight at 4°C and, then, collect the final sample (1mL).
Safety information
Chloroform is a CMR chemical reagent.
2d 3h 8m
RNA extraction using MasterPure Complete DNA & RNA purification kit.
2h
Extraction of 500µL viral nucleic acids using MasterPure Complete DNA & RNA Purification Kit (Epicentre).
Cellular lysis:
- Split in two tubes 250µL of viral sample : "tube 1" and "tube 2".
This two tubes will be pooled and the end of extraction for concentration.
- Thoroughly mix the several buffers to homogenize them before dispensing.
- Dilute 4μL of Proteinase K into 600μL of 2X T and C Lysis Solution.
- Vortex 10s.
- Add 250µL of 2X T and C Lysis Solution containing the Proteinase K to each tube of viral sample and mix thoroughly.
- Incubate at 65°C for 15 minutes in a Eppendorf thermomixer at 1000rpm with smartblock 1,5mL.
- Place the samples on ice for 5 minutes to inactivate the proteinase K.
Nucleic acids precipation and extraction :
- Add 250 μL of MPC Protein Precipitation Reagent to 500 μL of lysed sample.
- Vortex vigorously for 10 seconds.
- Centrifuge. 11000 x g, 12°C, 00:10:00
- Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
- Centrifuge. 11000 x g, 12°C, 00:10:00
- Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
- Add 840 μL of cold isopropanol to the recovered supernatant. Invert tubes 40 times.
- Centrifuge tubes to pellet the total nucleic acid. 20800 x g, 12°C, 00:10:00
- Carefully pour off the isopropanol without dislodging the pellet.
Contaminating DNA digestion with DNase :
- Prepare DNAse I solution (1/40) : 10µL Rnase Free Dnase I into 390µL Dnase Buffer.
- Completely resuspend the total nucleic acid pellet in 200 μL of DNase I solution (1/40).
- Incubate at 37°C for 10 minutes.
- Add 200 μL of 2X T and C Lysis Solution.
- Vortex for 5 seconds.
- Add 200 μL of MPC Protein Precipitation Reagent.
- Vortex 10 secondes.
- Place on ice for 4 minutes.
- Centrifuge 11000 x g, 12°C, 00:10:00 to pellet the debris.
- Transfer the supernatant containing the RNA into a clean microcentrifuge tube and discard the pellet.
- Centrifuge 11000 x g, 12°C, 00:03:00 keep and transfer the supernatant into a new tube.
- Add 840µL of cold isopropanol to the supernatant. Invert tubes 40 times.
- Centrifuge 20800 x g, 12°C, 00:10:00 and discard supernatant.
RNA precipitation:
- Rinse twice with cold 70% ethanol, being careful to not dislodge the pellet.
- Centrifuge. 11000 x g, 12°C, 00:00:30
- Remove all the ethanol by pipetting and keep at room temperature the tube open during 10 minutes for the ethanol evaporation .
- Resuspend the total nucleic acids (RNA) of "tube 1" in 35 μL of TE Buffer.
- Re use this solution for resuspend the total nucleic acids of the "tube 2" in the aim of concentrate sample.
- Keep 2µl for Qubit quantification and store RNA extract at -80°C.
2h
RNA Quantification using Qubit 4 fluorimeter.
40m
- Quantify the RNA concentration using the Qubit 4 fluorimeter and the RNA BR Assay kit which provides a detection range of 10 and 1200ng of RNA.
Retrotranscription to cDNA.
2h
By sample, add in 0,2 ml microtube :
- 1µL Random primers (250ng)
- 11µL RNA extract (Total RNA should range between 10pg - 5µg - complete with RNA-free water if necessary)
- 1µL 10mM dNTP
qsp 13µL : Add Nuclease Free water to complete.
Thermocycler parameter : 65°C 5min.
2min in ice.
In the same microtube, add :
- 4µL 5X First strand buffer
- 1µL 0,1M DTT
- 2µL Superscript III RT (400U/µl)
- 1µL RNAse OUT Recombinant Rnase Inhibitor.
Thermocycler parameter : 25°C 10min > 50°C 60min > 70°C 15min.
Store cDNA at -20°C.
cDNA Quantification using Qubit 4 fluorimeter.
Quantify the DNA concentration using the Qubit 4 fluorimeter and the dsDNA BR Assay kit which provides a detection range of 4 and 2000ng of DNA.
Protocol references
Arsenieff L, Simon N, Rigaut-Jalabert F, Le Gall F, Chaffron S, Corre E, Com E, Bigeard E and Baudoux A-C (2019) First Viruses Infecting the Marine Diatom Guinardia delicatula. Front. Microbiol. 9:3235. doi: 10.3389/fmicb.2018.03235
Keller, M. D., Seluin, R. C., Claus, W., and Guillard, R. R. L.Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23, 633–638. doi:10.1111/j.1529-8817.1987.tb04217