Mar 11, 2026
RNA extraction Using TRIzol
- Jadranka Nappi1
- 1The University of New South Wales, Kensington, NSW, Australia
Protocol Citation: Jadranka Nappi 2026. RNA extraction Using TRIzol. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwwq2zvmk/v1
Manuscript citation:
Mangott, A., et al. "Ulva lactuca as a functional ingredient and water bioremediator positively influences the hepatopancreas and water microbiota in the rearing of Litopenaeus vannamei." Algal Research 51 (2020): 102040.
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: March 11, 2026
Last Modified: March 11, 2026
Protocol Integer ID: 313114
Keywords: RNA, TRIzol, rna extraction, purification of total rna, using trizol, including crustacean gut tissue, marine sample, crustacean gut tissue, extraction, total rna, trizol this protocol, rna precipitation, based extraction, purification, cdna synthesis for downstream application, dnase treatment, cdna synthesis
Abstract
This protocol outlines the extraction and purification of total RNA from marine samples, including crustacean gut tissue, water filters, and surface swabs. Samples are homogenised and processed using TRIzol-based extraction, followed by phase separation, RNA precipitation, DNase treatment to remove genomic DNA, RNA re-purification, and cDNA synthesis for downstream applications such as PCR or sequencing.
Attachments
Materials
Reagents
TRIzol reagent (Invitrogen, Cat. No. 15596018)
Chloroform (Sigma, Cat. No. C2432-1L)
Isopropanol (molecular grade)
Ethanol 75% (molecular grade)
Molecular-grade water
RNA-grade glycogen
DNase I and 10× reaction buffer
DNase stop solution
3 M sodium acetate (pH 5.2)
Random hexamer mix
Protoscript II First Strand cDNA Synthesis Kit (NEB #E650S)
Consumables
1.5 mL microcentrifuge tubes (molecular grade)
0.2 mL PCR tubes
Lysing Matrix E bead tubes (MP Biomedicals, Cat. No. 116914100)
Aerosol barrier pipette tips (10 µL, 20 µL, 200 µL, 1000 µL)
Cleaning reagents
RNase AWAY
SDS/H₂O₂ RNase cleaning solution
10% bleach
70% ethanol
Equipment
Homogenisers (Sigma, Cat. No. Z359947-100EA)
Tissue Lyser or bead-beating homogeniser
Microcentrifuge capable of 13,000 × g at 4 °C
Heating block
Incubator or drying oven set at 37 °C
Fume hood
Qubit fluorometer
Nanodrop spectrophotometer
Gel electrophoresis system
Troubleshooting
Before start
Prepare:
13,000xg centrifuge, that goes to 4°C
Fume hood
Tissue Lyser
Heating block
Incubator/oven at 37°C for drying tubes
Homogenising samples + splitting into TRIzol or PowerSoil Methods
Homogenisation is performed to mechanically disrupt the sample and release nucleic acids. The homogenised material is then divided into two aliquots to allow parallel extraction workflows, with one portion processed for RNA extraction using TRIzol and the other reserved for DNA extraction using the PowerSoil method.
a) In a 1.5mL tube, homogenise sample (cut into sections first and grind with sterile pestle).
b) Add 100uL of MG water.
c) Split sample evenly into a second 1.5mL tube using a wide-orifice or cut pipette tip (~50uL each).
d) Place one tube in -20C storage for later PowerSoil extraction (DNA Only).
e) Rinse the remaining tube with 500uL TRIzol and transfer into lysis bead tube (Lysing Matrix E MP Bio).
*Note: Move to fumehood
f) Add remaining 500uL TRIzol into lysis tube.
g) Bead beat using Tissue lyser, 4x 30 sec with 30 sec pauses at maximum speed)
Phase separation
During phase separation, chloroform is added to the TRIzol lysate and centrifuged to separate the mixture into an aqueous phase containing RNA, an interphase containing DNA, and an organic phase containing proteins and lipids.
a) Transfer from lysis tube to 1.5 ml microcentrifuge tube, avoiding the beads.
b) Vortex for 30s, rest for 30s and vortex again.
c) Incubate at RT for 5 min.
*Note: It’s possible to freeze samples at -20°C.
d) Add 200 µl of room temperature chloroform. Mix by inverting the tube several times.
e) Incubate at RT for 2-3 min.
f) Centrifuge for 15 minutes at 12,000 × g at 4°C.
Transfer the upper aqueous phase containing the RNA to a new tube.
RNA isolation
RNA isolation using TRIzol involves cell lysis in a phenol–guanidine reagent followed by phase separation with chloroform, precipitation of RNA with isopropanol, and washing with ethanol to obtain purified RNA suitable for downstream molecular analyses.
RNA precipitation
a) Add 1 µl of glycogen (20 µg/µl; 20 µg added) to the upper aqueous phase.
b) Add 600 µl of cold isopropanol, mix by inversion.
c) Incubate for 10 min at RT.
d) Centrifuge for 10 minutes at 12,000 × g at 4°C.
e) Discard the supernatant with a pipette.
f) Add 1000 µl of 75% ethanol.
g) Mix by gently tapping the tube and inverting the tube to clean tube wall.
Note: RNA can be stored in 75% ethanol for up to 1 week at 4°C.
h) Spin at 12,000 xg, 5 min, 4°C.
i) Repeat wash step.
j) Discard the supernatant with a pipette.
Can stop here: RNA in minimum amount of ethanol for cold shipping
k) Dry the pellets by leaving the tubes opened at 37°C in oven for ethanol evaporation for up to 20 min.
l) Add 30 µl of water (RNase-free).
m) Incubate at 55°C for 5-10 min, tapping the tubes during the process.
DNase treatment
a) For each reaction:
| Single Reaction | ||
| RNA (µL) | 30 | |
| Dnase (µL)* | 1 | |
| 10x reaction buffer (µL)* | 4 | |
| MG Water* | 5 | |
| total (µL) | 40 |
b) *Add as master mix (10µl each sample) of Dnase/buffer/water.
c) Mix gently by pipetting. DO NOT vortex, spin briefly if necessary.
d) Incubate at 37°C for 30 min.
e) Inactivate DNase I by adding 4 µl of Stop Solution, mix gently and incubate tubes at 65°C for 10 min (44µL).
f) Store RNA samples at -80°C, if not performing next step.
RNA re-purification
*Note a)-c) Do not add as master mix
a) Add 4µl of 3 M sodium acetate (pH 5.2, ice cold). Mix well by tapping the tube.
b) Add 1 µl of glycogen (20 µg/µl; 20 µg added). – optional: use glycogen when you expect low yield (49uL).
c) Add 50 µl of isopropanol (ice-cold). Mix well by tapping the tube.
d) Incubate samples at -80°C for at least 1h (or at -20°C overnight).
e) Spin at 12,000 xg, 10 min, 4°C.
f) Remove supernatant with a pipette.
g) Add 1000 µl of 75% ethanol.
h) Mix by gently tapping the tube and inverting the tube to clean tube wall.
i) Spin at 12,000 xg, 5 min, 4°C.
j) Repeat washing step.
k) Dry the pellets by leaving tubes opened at 37°C for ethanol evaporation.
l) Add 20 µl of water (MG).
m) Incubate at 55°C for 5-10 min.
n) Check RNA concentration using Qubit, and quality on gel and Nanodrop (aliquot 5uL).
o) Store RNA samples at -80°C.
cDNA synthesis – Protoscript II (NEB #E650S)
1. In a 0.2mL PCR tube, add 6µL RNA and 2µL random hexamer mix
2. Incubate 65°C for 5 minutes (PCR machine)
3. Add 10µL reaction mix, 2µL enzyme mix.
4. Incubate (PCR machine) which entails:
25°C – 5 minutes
42°C – 60 minutes
80°C – 5 minutes
5. Clean for 16S PCR or just dilute before 16S PCR.