Protocol Citation: Dakota Betz 2026. RNA Extraction with Trizol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm1zxbv3p/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 04, 2026
Last Modified: February 04, 2026
Protocol Integer ID: 242560
Keywords: protocol for rna extraction, rna extraction, using trizol, trizol, rna, extraction, rna extraction with trizol
Abstract
This is a protocol for RNA extractions (using Trizol). A maximum of 3 samples can be extracted at once using this protocol. The entire protocol should be run in the fumehood. This protocol is time- and temperature-sensitive, so read all instructions before attempting and make sure you are adequately prepared.
Guidelines
A maximum of 3 samples can be extracted at once using this protocol.
Materials
- RNase Away
- RNA free tubes (1.6 and 2 ul)
- Trizol
- 100% ethanol (new every time)
- RNase free water (new every time)
- Direct-zol RNA miniprep kit (Zymo research) <- contains all other reagents needed
Protocol materials
UltraPure ™ Distilled Water
Chloroform
Safety warnings
Trizol is BAD--conduct entire extraction protocol in the fume hood! You will need to move the centrifuge, bead basher, and other equipment into the hood before starting. Make sure you have icepacks available as this protocol is time- and temperature-sensitive.
Before start
Celean all surfaces, tools, and gloves with RNase Away. Don't forget to clean pipettes, plastic pipettes, ice blocks, centrifuge, etc.
Make sure buffers have been prepped if it's a new kit (instructions in kit for which reagents/how much to add to each).
RNA purification & HAV
1h 5m 30s
Add 200 µL of virus susoension to be extracted to a 1.5 mL tube
Lysis
Add 800 µL of UltraPure ™ Distilled Water to tube (1 per sample). Mix thoroughly by brief pulse vortexing and incubate for 00:05:00 at room temperature.
5m
Phase Separation
Add 160 µL of Chloroform to each tube. Shake vigorously for 00:00:30 , then incubate for 00:02:00 to 00:03:00 at Room temperature .
5m 30s
Centrifugation
Centrifuge samples 12000 rpm, 4°C, 00:15:00
15m
Aqueous Phase Collection
After centrifugation, the mixture separates into lower red, phenol chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Carefully remove 480 µL of the clear upper aqueous phase and transfer it to a new, labeled microcentrifuge tub0 °C e. Please note: Extract 80% of the aqueous RNA layer leaving 20% behind in the tube to prevent contaminating the aqueous layer.
RNA Precipitation
Add 400 µL of UltraPure ™ Distilled Water and 1 µL of UltraPure ™ Distilled Water co-precipitant to the aqueous phase. Mix by tilting 10 times and incub800 µL ate for 00:20:00 at −20 °C .
20m
RNA Pelleting
Centrifuge for 12000 rpm, 4°C, 00:10:00
10m
Ethanol Wash
A dark blue RNA pellet should be visible at the bottom of the tube if GlycoBlue was used.
Carefully discard the supernatant and wash the pellet with 800 µL of 75 % (v/v) Ethanol
Wash Centrifugation
Centrifuge for 12000 rpm, 4°C, 00:05:00
5m
Ethanol Removal
Discard the supernatant. Briefly centrifuge again to collect residual ethanol, then use a P200 pipette tip to remove the remaining ethanol without disturbing the pellet. Vacuum or air dry the RNA pellet for 00:05:00 . It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility.
5m
- RNA Resuspension Resuspend the RNA pellet in 50 µL of UltraPure ™ Distilled Water by vortexing. The dilution step was intentionally omitted to maximize sensitivity.
10) Store everything at -80 °C