Feb 19, 2025
Extraction of RNA from soybean leaves using the trizol method.
- Ian de Paula Alves Pinto1,
- Phedra Gusmão da Silva de Oliveira1
- 1Universidade Federal de Viçosa
Protocol Citation: Ian de Paula Alves Pinto, Phedra Gusmão da Silva de Oliveira 2025. Extraction of RNA from soybean leaves using the trizol method.. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32851v25/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: June 29, 2024
Last Modified: February 19, 2025
Protocol Integer ID: 102592
Keywords: RNA, soybeans
Abstract
The RNA extraction protocol from soybean leaves using the Trizol method presented below is an adaptation of the traditional method, with adjustments to enhance efficiency and minimize contamination and nucleic acid degradation, ensuring an optimized execution.
Guidelines
Guidelines for Performing the Experiment
RNase-Free Environment
The experiment must be conducted in an RNase-free environment to prevent RNA degradation.
All materials used must be pre-autoclaved.
Work surfaces should be cleaned with 10% SDS prior to the experiment.
Sample Maintenance
During the experiment, samples must be kept in a container with ice to preserve RNA integrity.
Quality Control
Extracted samples should undergo quality control using spectrophotometry.
RNA quality must be confirmed by electrophoresis on a 1% agarose gel.These steps ensure RNA integrity and the reliability of subsequent procedures.
Materials
Trizol TRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
Ethanol 75%
Isopropanol
Chloroform
Microtube 1.5 mL
Centrifuge with temperature control
Mortar and pestle
Safety warnings
As Trizol is a toxic reagent, the entire experiment must be carried out in an exhaust hood and with gloves on.
Ethics statement
No animals are used in the protocol.
Before start
To carry out the protocols, the microtubes, tips, mortars and pistils must be autoclaved beforehand. In addition, the benches and pipettors must be previously cleaned with 10% SDS and the protocol must be carried out in an exhaust hood.
Extraction of RNA from soybean leaves using the trizol method.
Extraction of RNA from soybean leaves using the trizol method.
39m
39m
Grind 100 mg of soybean leaves in liquid nitrogen in an autoclaved mortar.
Pass the mixture into a previously autoclaved 1.5 mL microtube and add 1 mL of TriZol.
Homogenize the sample for 5 minutes and keep it in a container with ice.
5m
Add 200µL of chloroform to the sample and mix for 3 minutes.
3m
Centrifuge for 15 minutes at 12000 rpm 4ºC.
15m
Pipette off 400 µL of the supernatant and add it to a new tube.
Add 300 µL of ice-cold isopropanol and mix for 1 minute. Then leave the samples at room temperature for 10 minutes.
11m
Centrifuge for 10 minutes at 12000 rpm 4ºC.
Discard the supernatant and add 1 mL of ice-cold 75% ethanol, keeping the microtubes on ice.
Centrifuge for 5 minutes at 9500 rpm 4ºC.
5m
Repeat the previous two steps 2 times.
Dry the pellet at room temperature until there is no more ethanol inside the microtube.
Dilute the pellet in 35 µL of rnase-free water or water for injections followed by treatment with dnase.
Acknowledgements
I would like to thank the Federal University of Viçosa and the Protein and Peptide Biochemistry Laboratory