Jun 13, 2025
RNA Extraction with Trizol
- Dakota Betz1
- 1ucsd
- Rouse Lab
Protocol Citation: Dakota Betz 2025. RNA Extraction with Trizol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrop22vmk/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: March 04, 2020
Last Modified: June 13, 2025
Protocol Integer ID: 33758
Keywords: protocol for rna extraction, rna extraction, using trizol, trizol, rna, extraction
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)
- Petri dishes
- Trizol
- 100% ethanol (new every time)
- RNase free water (new every time)
- Direct-zol RNA miniprep kit (Zymo research) <- contains all other reagents needed
Troubleshooting
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).
Homogenize Tissues
Add 770 uL of Trizol to bead bashing tube (1 per sample).
Pour tissue into a clean Petri dish, keep on cooling block (flat ice pack).
Transfer pieces of tissue to bead bashing tube, blot RNAlater with a kim tissue.
May be a very small amount of tissue (if so use all) and blot until it’s REALLY dry. Do all of this on top of an ice pack to keep it cold.
Beadbash 3-4 times for 15 sec at max speed. Cool down 5-10 seconds in between, placing tube in/on ice block. Stop as soon as tissue is dissolved!
Transfer liquid to a 1.6 mL RNase free tube and centrifuge 12000 x g, 00:02:00 . Use block from lab freezer to keep tubes cold.
Transfer supernatant to a new 1.6 mL RNase free tube.
RNA purification & DNase I treatment
Add 700 uL of 100% ethanol to each tube. Vortex.
Load 700 uL into ZymoSpin IIC Column in the 2mL-size RNase-free collection tube. Use this size for all remaining steps. Centrifuge 12000 x g, 00:01:00 .
Note
For sea cucumbers, urchins, or other tissues that might clog the filter, split them into two ZymoSpin IIC Columns instead of doing step 9: i.e., treat like two "separate" samples for the rest of the protocol, then combine them at the end.
1) Discard flow-through. Repeat with remaining 700 uL (if applicable). If first half did not flow through completely, centrifuge12000 x g, 00:02:00 .
2) Transfer column to RNase-free tube and do in-column digestion. Wash with 400 uL of RNA Wash Buffer. Centrifuge 12000 x g, 00:00:30 , discard flow-through.
3) Add 80 uL of DNase I Reaction Mix (75 µL DNA digestion Buffer (kit), 5 µL DNase I (freezer, green cap); scaled) directly to the column. Incubate for 00:15:00 at room temperature . Centrifuge 12000 x g, 00:00:30 .
4) Add 400 uL of Direct-zol RNA PreWash. Centrifuge 12000 x g, 00:01:00 .
5) Discard flow through. Repeat previous step. (Pre-wash and centrifuge again).
6) Add 700 uL of RNA Wash Buffer. Centrifuge 12000 x g, 00:01:00 . Discard flow-through. Centrifuge again in emptied collection tube 12000 x g, 00:02:00 .
7) Transfer column to labeled RNase free tube.
8) Add 26 uL of RNase-free water to column. Incubate 00:03:00 at room temperature . Centrifuge 12000 x g, 00:01:00 .
9) Combine tubes if applicable (see note on step 8). Take 5 uL for QC/Qubit. The kit is called “Qubit RNA HS” and has weirdly clear dye and different standards.
10) Store everything at -80 °C