May 06, 2026
Phenol-free RNA extraction of Rubus armeniacus berries
- Katharina Wolff1,1,
- Boas Pucker1,1
- 1Plant Biotechnology and Bioinformatics, IZMB, University of Bonn
Protocol Citation: Katharina Wolff, Boas Pucker 2026. Phenol-free RNA extraction of Rubus armeniacus berries. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwwr4dvmk/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 have used this protocols successfully for Rubus armeniacus berries
Created: April 30, 2026
Last Modified: May 06, 2026
Protocol Integer ID: 316038
Keywords: RNA extraction, berry, free rna extraction of rubus armeniacus, berries of rubus armeniacus, extraction of rna, free rna extraction, berry, rna, rubus armeniacus, extraction, phenol
Abstract
Protocol optimized for the extraction of RNA out of berries of Rubus armeniacus.
Materials
Materials
Materials: mortar & pestle, water bath, centrifuge for 2 ml tubes with cooling function, NanoDrop
RNA Extraction Buffer
300 millimolar (mM) TRIS HCL (pH 8.0)
25 millimolar (mM) EDTA (pH 8.0)
2 Molarity (M) NaCl
2 Mass / % volume CTAB
2 Mass / % volume PVPP
2 % (v/v) β-mercaptoethanol
β-mercaptoethanol is added directly prior to extraction
TE Buffer
10 millimolar (mM) TRIS (pH 8.0)
0.1 millimolar (mM) EDTA (pH 8.0)
This Buffer needs to be RNAse free
Choloroform:Isoamylalkohol (24:1)
10 M LiCl
Ethanol (abs.)
70% Ethanol (ice cold)
N2
Safety warnings
ß-ME is a neurotoxin. Use gloves when handling it.
Perform all chloroform procedures under the fume hood.
Before start
This protocol is optimized for the extraction of RNA from berries of Rubus armeniacus and has been successfully applied to berry tissue from different ripening stages
day 1
For each berry prepare 2 x 2mL reaction tube with 2% β-mercaptoethanol in900 µL of Extraction Buffer (make sure the buffer has been mixed as PVPP is not soluble) and preheat at 65 °C
Homogenize 1 frozen berry with mortar and pestle in liquid N2
Add homogenized material (powder) to the two tubes of preheated extraction buffer and resuspend
carefully
It is crucial to completely resuspend the powder. Avoid transfer of ice.)
Incubate at 65 °C for00:20:00 and invert tubes frequently to
carefully mix the solution.
Centrifuge the samples at11000 x g, 4°C, 00:10:00
Transfer the supernatant to fresh 2 mL reaction tubes
Add an equal volume of Chloroform:Isoamylalcohol (24:1) to the tubes and centrifuge at 12000 x g, 4°C, 00:10:00
Transfer upper phase into a fresh tube and repeat the extraction with an equal volume of Chloroform:Isoamylalcohol (24:1) and centrifuge 12000 x g, 4°C, 00:10:00
Transfer the upper phase to a clean 1.5 mL reaction tube and add 1/4 volume of 10 M LiCl and incubate the sample overnight at 4 °C
day 2
Centrifuge the samples 12000 x g, 4°C, 00:20:00
Discard supernatant and wash pellet with 500 µL 70% ice cold ethanol
Centrifuge 12000 x g, 4°C, 00:05:00
Decant ethanol and dissolve RNA pellet in 100 µL TE buffer
Add 300 µL ice cold ethanol (abs) and gently mix by inversion
Centrifuge at12000 x g, 4°C, 00:05:00
Discard supernatant and dissolve RNA pellet in 20 µL of TE buffer
Perform quality checks of RNA such as NanoDrop measurements and Gel electrophoresis and store at -80 °C
Protocol references
This RNA extraction has been based on the methods of :
Wang, K., Liu, M., Cui, F., Asiegbu, F., 2021. A simple phenol-free isolation method for high-quality RNA from bilberry. MethodsX 8, 101481. https://doi.org/10.1016/j.mex.2021.101481
Reid, K.E., Olsson, N., Schlosser, J., Peng, F., Lund, S.T., 2006. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol. 6, 27. https://doi.org/10.1186/1471-2229-6-27