Jul 31, 2025
RNA and Organic Reagent Extraction for Analysis
This protocol is a draft, published without a DOI.
- Andrew Reed1
- 1University of Tennessee Knoxville
- Andrew Reed: Primary author and validator
- CASB methods
Protocol Citation: Andrew Reed 2025. RNA and Organic Reagent Extraction for Analysis. protocols.io https://protocols.io/view/rna-and-organic-reagent-extraction-for-analysis-g5x8by7rx
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: July 21, 2025
Last Modified: July 31, 2025
Protocol Integer ID: 222944
Keywords: RNA, Carotenoids, RNA extraciton, Organic Extraction, Plant, Plant cell, CTAB, extraction of rna, organic reagent extraction for analysis, organic reagent extraction, extraction of organic compound, rna yeild, expected rna yeild, rna, extraction, carotenoid, organic compound, single sample, μl per sample
Disclaimer
This is my first upload to protocols.io I would appreciate any help improving this protocol for this platform.
The RNA extraction from this protocol has been extensively used in my lab to obtain high-yeild, high-purity RNA samples from plant tissue for a while now and I thought it would be good to share it with the wider world.
Mostly because Zymogen's lysis buffer sucks for plant cells and typically cuts the yield and purity from samples in half compared to lysis with CTAB, but going through an entire chloroform extraction takes far to long and working with chloroform can be troublesome. While chloroform is used in this protocol that is mostly to extract the organic compounds and can be ignored if you only want to extract RNA.
Abstract
This protocol is for the extraction of RNA and organic compounds (primarily carotenoids) from a single sample. Expected RNA yeilds should lie around 500-1000ng/μL per sample. Extraction of organic compounds will vary by solvent used.
Guidelines
Separation of the aqueous and organic phases should be done at the same time, and the organic phase can be left open in a fume hood while the RNA purification steps are completed.
Organic compounds extracted will vary by solvent used after chloroform evaporation.
Materials
Extraction Buffers and kits:
P1 Solution:
- 2 mg of canthaxanthin (internal standard)
- 20 mL chloroform
CTAB Extraction Buffer (for 600mL):
- CTAB: 12g
- PVP: 6g
- Tris-HCl: 9.456g -OR- Tris-HCl stock solution (1.5M, pH 8.0): 40 mL
- NaCl: 49.09g
- EDTA: 3.507g -OR- EDTA stock solution (0.5M, pH 8.0): 24 mL
- Add H₂O up to 600mL
- Autoclave -OR- Heat up to dissolve and 0.22μm filter to sterilize.
- Aliquot in 15mL conical tubes and store at -20°C.
Troubleshooting
Safety warnings
Steps of this procedure involve the use of chloroform and should be done in a fume hood to avoid harm.
Before start
Before procedure set water bath at 55-65°C.
Tissue Lysis
17m 30s
Homogenize 25 mg of frozen tissue sample in liquid nitrogen using tissue lyser (we tested two punches of leaf discs from 2mL tubes).
30s
Add 600μL pre-warmed Extraction Buffer (55-65°C) to each sample.
Vortex immediately and place in the water bath. Incubate the samples in the water bath for at least 10 minutes and vortex after incubation.
10m
Centrifuge at maximum speed (17,000xg) for 5 minutes and transfer all of the supernatant to a clean 2mL Eppendorf tube.
5m
Add 560μL of chloroform and 40μL P1 Solution, then vortex for 30 seconds and centrifuge at maximum speed (17,000xg) for 2 minutes.
2m
(if only RNA is desired then skip the addition of chloroform and move directly to RNA extraction as the separation of RNA is achieved with spin columns).
RNA extraction
25m 30s
Transfer aqueous (upper) phase to a Zymogen Spin-Away Filter (yellow) in a collection tube without disturbing the white interphase.
Centrifuge at 11,000xg for 30 seconds and save the flow-through to remove a majority of genomic DNA.
30s
Add an equal volume of ethanol (95-100%) to the flow-through and mix well. Load 750-800μL mixture onto the Zymo-Spin IIICG Column (green) and spin down at 11,000xg for 30 seconds. Discard the flow-through. Repeat until all the samples are loaded on the columns.
30s
Wash with 400μL RNA wash buffer and centrifuge (11,000xg for 30 seconds). Discard the flow-through.
30s
Spin the columns at 11,000xg for 30 seconds to remove residual buffer to dry the columns.
30s
Prepare DNase I Enzyme mixture by mixing 75μL of DNase I buffer with 5μL of DNase I.
Add 80μL of DNase I mixture to each column and let reaction sit for 15 minutes at room temperature (20-30°C).
15m
Add 400μL RNA Prep Buffer to the column and centrifuge (11,000xg for 30 seconds). Discard the flow-through.
30s
Add 700μL RNA Wash Buffer to the column and centrifuge (11,000xg for 30 seconds). Discard the flow-through.
30s
Add 400μL RNA Wash Buffer to the column and centrifuge (11,000xg for 30 seconds). Discard the flow-through.
30s
Centrifuge one additional time to dry column (11,000xg for 1 minute), and carefully remove the columns from centrifuge.
1m
Add 30-50μL DNase/RNase-Free Water directly to column matrix and wait for 5 minutes.
5m
Centrifuge at 11,000xg for 1 minute to recover the RNA samples.
1m
Snap freeze RNA samples in liquid nitrogen and store at -80 after nanodrop quantification.
Organic extraction
1d 0h 12m 30s
Collect the organic (lower) phase into 2mL Eppendorf tubes.
When removing the organic phase some air should be let into the solution to remove any of the remaining aqueous phase from the tip of the pipette and avoid nucleotide contamination. The pipette tip should also be tapped on the side of the tube after it is removed to ensure no aqueous phase makes it into the final tube.
Allow the solution to evaporate (either in a SpeedVac or in fume hood overnight).
1d
Resuspend the pellet in 300μL acetone and sonicate until fully dissolved (three 30 second intervals at 50 amps with 1 min pause between each interval).
2m 30s
Centrifuge at 12,000xg for 5 min.
5m
Move the supernatant into a clean tube and repeat the centrifugation process.
5m
Remove supernatant and filter through a 0.2μm membrane (PTFE) and inject 33μL into the HPLC.
HPLC METHOD
50m
Set up the HPLC system using the following parameters:
Method name: MP-ISO-NOREF-FLR (exact variant depends on column condition)
- Column: YMC C30 Carotenoid, 250 × 4.6 mm, 3 μm
- Flow rate: 1 mL min-1
- Column temperature: 25 °C
Prepare mobile phases:
- A: Methanol
- B: H₂O : Methanol : Ammonium acetate (80 : 20 : 0.1 %)
- C: tert-Butyl methyl ether
Set the gradient program as follows:
| Time (min) | A% | B% | C% | |
| 30.0 | 30 | 5 | 0 | |
| 12.1 | 80 | 5 | 0 | |
| 0.0 | 95 | 5 | 0 | |
| 12.0 | 95 | 5 | 65 | |
| 30.1 | 95 | 5 | 0 | |
| 50.0 | 95 | 5 | 0 |
50m
Set detection parameters:
- Diode-array:
- 290 nm (Phytoene)
- 472 nm (Carotenoids)
- 650 nm (Chlorophylls)
- Fluorescence:
- Ex = 290 nm / Em = 330 nm (Tocopherols)
Protocol references
RNA extraction:
Quick-RNA Miniprep (Zymo Research Inc., R1055)
Carotenoid extraction and analysis:
Andersen, Trine B., et al. "An engineered extraplastidial pathway for carotenoid biofortification of leaves." Plant Biotechnology Journal 19.5 (2021): 1008-1021.
Acknowledgements
I would like to aknowledge Dr. Yongil Yang and Dr. Li Li for their contributions in helping for the validation of this protocol. Additionally I would like to aknowledge the members of the CASB at the University of Tennessee Knoxville for their contributions by further validating this protocol through its continued use.