Feb 26, 2026
RNA extraction for Kryptoperidinium triquetrum CCAP 1116/3 and Nitzschia laevis UTEX_B_2047
- Will Lewis1,
- Ross F. Waller1
- 1Department of Biochemistry, University of Cambridge
Protocol Citation: Will Lewis, Ross F. Waller 2026. RNA extraction for Kryptoperidinium triquetrum CCAP 1116/3 and Nitzschia laevis UTEX_B_2047. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnm67vmk/v1
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: February 03, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 242538
Keywords: rna extraction, rna, TRIzol, Direct-zol, rna extraction for kryptoperidinium triquetrum ccap, rna extraction from kryptoperidinium triquetrum ccap, zol rna kit in this protocol, zol rna kit, rna extraction, kryptoperidinium triquetrum ccap, standard trizol phase separation by centrifugation, combining standard trizol phase separation, trizol, addition of trizol, rna, extraction, cell harvesting
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: GBMF9194
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Abstract
A methodology for cell harvesting and RNA extraction from Kryptoperidinium triquetrum CCAP 1116/3 and Nitzschia laevis UTEX_B_2047 for the purposes of RNA-seq.
The cells of these two species have tough cell walls, which prevent them lysing from the addition of TRIzol alone. Therefore, they require mechanical lysis using a pestle.
Combining standard TRIzol phase separation by centrifugation with the Zymo Research Direct-zol RNA Kit in this protocol improves sample purity far greater than using either of these two methods on their own.
Materials
A Direct-zol RNA Miniprep Kit
TRIzol
Chloroform
Isopropanol
Ethanol
10 µl, 100 µl and 1000 µl pipettes and corresponding sterile RNase-free filter tips
50 ml centrifuge tubes
1.5 ml Eppendorf DNA LoBind Tubes
Sterile disposable pestles compatible with 1.5 mL tubes.
Tube racks
Invitrogen UltraPure DNase/RNase-Free Distilled Water
Centrifuge with adapter for 1.5 ml tubes
Troubleshooting
Safety warnings
Read MSDS for all reagents prior to performing protocol and follow appropriate safety precautions. Perform all steps involving opening a tube or container containing TRIzol and chloroform in a fume cupboard wearing appropriate PPE, and be sure to collect and dispose of liquids and consumables contaminated with these reagents according to the department's waste disposal procedures.
Before start
Cool centrifuges to 4°C.
Cell lysis and phase separation
25m
Take 50 µl aliquot into a sterile 1.5 mL tube that is suitable for use with a pestle grinder. Store remaining 250 µL sample at -20 °C to be processed as needed in the future.
Pestle grind 50 µl sample for 1 minute to lyse the cells.
Add 750 µl TRIzol to the lysed sample and mix by pipetting up and down.
Pellet cell fragments by centrifugation at 12,000 x g for 10 minutes at 4 °C and transfer the supernatant to a new sterile tube.
Add 0.25 volumes of chloroform per volume of sample (measure sample volume using a pipette) and thoroughly mix by vortexing for 15 seconds.
Incubate at room temperature for 10 minutes
Centrifuge at 12,000 x g for 15 minutes at 4 °C for phase separation
Remove the upper RNA-containing aqueous phase of the sample after phase separation and transfer this to a new sterile tube.
Make sure not to pipette too close to the interface to avoid contaminating the sample with DNA. Only very small quantities of RNA are needed for RNA-seq, so no need to try and collect the total aqueous phase, as this will decrease sample quality.
Gently add 0.6 volumes of ethanol to aqueous phase and mix by gentle inversion
Direct-zol™ RNA Miniprep Kit (Zymo Research)
The protocol steps in this section have been directly adapted from the manufacturer's protocol for the Direct-zol RNA Miniprep kit:
Transfer the ethanol and aqueous phase mixture into a Zymo-Spin IICR Column in a Collection Tube and centrifuge at 15 000 x g for 30 seconds. Transfer the column into a new collection tube and discard the flow-through.
DNase I treatment
Add 400 μl RNA Wash Buffer to the column and centrifuge at 15 000 x g for 30 seconds.
In an RNase-free tube (not included), add 5 μl DNase I (6 U/μl)*, 75 μl DNA Digestion Buffer and mix by gentle inversion. Add the mix directly to the column matrix.
Incubate at room temperature (20-30°C) for 15 minutes. Proceed to next step.
Add 400 μl Direct-zol RNA PreWash to the column and centrifuge at 15 000 x g for 30 seconds. Discard the flow-through and repeat this step.
Add 700 μl RNA Wash Buffer5 to the column and centrifuge at 15 000 x g for for 1 minute to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
To elute RNA, add 40 μl of DNase/RNase-Free Water directly to the column matrix and centrifuge.
Quantification, quality control and storage
25m
Analyse samples using Nanodrop to ensure the 260/280 ratio is ~2.0 and the 260/230 ratio is between 2.0 and 2.2.
Measure concentration of RNA and DNA using Qubit BR assay kits.
Store sample at -20°C