Jun 23, 2019
RNA extractions from de-etiolated Arabidopsis seedlings using CTAB
- 1The Hebrew University of Jerusalem;
- 2The Australian National University;
- 3University of Pennsylvania
- Pogson Group
- EBL_ANU
Protocol Citation: Akila Wijerathna Yapa, Andrew F Bowerman, Diep R Ganguly 2019. RNA extractions from de-etiolated Arabidopsis seedlings using CTAB. protocols.io https://dx.doi.org/10.17504/protocols.io.3f6gjre
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 in our group and it is working, though it will be continually developed.
Created: May 28, 2019
Last Modified: June 23, 2019
Protocol Integer ID: 23774
Keywords: RNA, de-etiolated seedlings, Arabidopsis, Plant
Abstract
A CTAB based method for extracting RNA, which is particularly useful for tough tissues. This has been adapted from a protocol that was originally used for pine tree tissue, which is difficult due to the high concentrations of polysaccharides, phenolics, and RNase (Chang, S., Puryear, J. & Cairney, J. Plant Mol Biol Rep (1993) 11: 113. https://doi.org/10.1007/BF02670468). The protocol described herein was an effective alternative to TRIzol based extractions for recovering RNA from juvenile de-etiolated tissues.
Guidelines
Ensure that you use RNA friendly practices (e.g. clean surfaces with RNAesy or 80% ethanol, use RNAase-free filter tips, use DEPC-treated water for buffers, adjust RNA buffer pH to be slightly acidic [e.g. ~6] in which RNA is more stable).
Materials
MATERIALS
Isoamylalcohol
Beta-mercaptoethanol
Chloroform
DEPC
Hexadecyltrimethylammonium bromide (CTAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #H9151
80% Ethanol
Lithium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #793620
Polyvinylpyrrolidone K 30Merck MilliporeSigma (Sigma-Aldrich)Catalog #81420
Extraction buffer
- 2% CTAB
- 2% PVP K30
- 100 mM Tris-HCl (pH 8.0)
- 25 mM EDTA (pH 8.0)
5. 2 M NaCl
6. Autoclave
7. 2% 2-mercaptoethanol (add before use = 100 uL per 5 mL buffer)
Safety warnings
Perform steps with 2-mercaptoethanol in a fume hood.
Before start
Prepare extraction buffer (requires autoclaving) and lithium chloride solution before starting. You may consider filter-sterilizing these to be extra cautious.
Harvest tissues into liquid nitrogen and grind (under liquid nitrogen) using mortar and pestle to obtain a fine powder. Ground tissue should be kept in safe-lock tubes in liquid nitrogen (or returned to -80C storage) until all samples are processed. This step is critical for efficient extractions so take your time here.
Add 1 mL of prewarmed (65°C) extraction buffer to each tube, mix well and incubate for 5 mins at 65°C (can be longer if needed e.g. 10-15 mins)
Add 200 ul of chloroform:IAA (24:1), mix well and spin @ 14,000 rcf for 10 mins. Remove upper aqueous phase to a new tube. Make sure not to disturb or pipette any material from the interface. Repeat chloroform:IAA step twice, being increasingly conservative when recovering the aqueous phase.
Add equal volume of 5 M LiCl to aqueous layer, mix well and incubate overnight @ -20°C.
Spin tubes @ 14,000 rcf for 20 mins @ 4°C.
Remove supernatant with pipette, add 1 mL 80% ethanol and invert tube ~10X. Centrifuge @ 7,500 rcf for 5 minutes @ RT. Remove ethanol and repeat.
Remove as much ethanol as possible using a pipette. Air-dry tubes with lid open for 1 min. Resuspend in 30 - 50 ul (depending on expected concentration and purpose, e.g. we aim for ~ 500 ng / ul) DEPC-treated water or low EDTA TE buffer (0.1 mM EDTA, 10 mM Tris base, pH 6.5).
Check quality of RNA e.g. visualize ~50 ng RNA on a 1% agarose gel or use BioAnalyser / LabChip GXII.