Optimized Protocol for High-Quality RNA Extraction from Grape Berry Skins Using Sorbitol Pre-Wash
Abstract
1. Introduction
2. Results
2.1. Enhancing RNA Extraction from Grape Berry Skins: The Impact of a Sorbitol Pre-Wash on Yield, Purity, and Integrity
2.2. Validation of RNA Extraction Protocol Through High-Throughput Analysis
2.3. Differential Impact of Sorbitol Pre-Wash on RNA Extraction from Different Grape Tissues
3. Discussion
4. Materials and Methods
4.1. Step-by-Step RNA Extraction Protocol with Sorbitol Pre-Wash
4.1.1. Sample Preparation and Sorbitol Pre-Wash
- Grind 250 mg (for non-commercial procedure) or 100 mg (for kit-based extraction) of frozen grape berry skins or other tissues into fine powder using liquid nitrogen.
- Divide samples into two groups:
- Without sorbitol pre-wash;
- With sorbitol pre-wash.
- Prepare the pre-wash buffer: 100 mM Tris-HCl (pH 8.0), 0.35 M sorbitol, 5 mM EDTA (pH 8.0), 1% PVP-40, and 1% β-mercaptoethanol (added fresh before use).
- Add 1.5 mL of pre-wash buffer to macerated tissue.
- Vortex vigorously and centrifuge at 2500× g for 5 min at 4 °C.
- Discard the supernatant containing polysaccharides and polyphenols.
- Repeat steps 2 to 4.
4.1.2. RNA Extraction with Non-Commercial Protocol (3 Days) (Modified from Rerie’s Protocol [31])
- Add 750 μL of extraction buffer (Tris-HCl 1M/SDS 1%/β-mercaptoethanol 5% + PVP 0.8%) to the sample.
- Vortex vigorously for 1 min.
- Add 1 mL of chloroform/isoamyl alcohol (24:1), mix well, and centrifuge.
- Recover the aqueous phase and precipitate RNA with 80 μL of KCl 2M and then with 330 μL of LiCl 8M.
- Store at −20 °C overnight.
- 6.
- Centrifuge at 12,000× g for 20 min at 4 °C.
- 7.
- Wash the RNA pellet multiple times with cold 2M LiCl.
- 8.
- Resuspend in 1X TE buffer and centrifuge at 12,000× g for 10 min at 4 °C.
- 9.
- Recover the supernatant and precipitate using 25 μL of 2M potassium acetate (pH 5.5).
- 10.
- Incubate 15 min on ice and centrifuge at 12,000× g for 15 min at 4 °C.
- 11.
- Recover the supernatant and precipitate with 1/10 of the volume of 3M sodium acetate (pH 6) and 2.5 volumes of ice-cold 100% ethanol.
- 12.
- Store at −20 °C overnight.
- 13.
- Collect RNA by centrifugation.
- 14.
- Wash the pellet with 1 ml of ice-cold 70% ethanol and resuspend in 30 μL of DEPC-treated water.
- 15.
- Perform DNase I (Sigma-Aldrich, St. Louis, MO, USA) treatment to remove DNA contamination.
4.1.3. RNA Extraction with Kit Protocol (Kit Plant/Fungi Total RNA Purification Kit from Norgen Biotek, Thorold, ON, Canada) (1 h)
- Lysate Preparation
- Transfer the powder to a 2 mL centrifuge tube and add 600 μL of Lysis Buffer C. Vortex vigorously.
- Incubate at 55 °C for 5 min. Mix the lysate 2 or 3 times during incubation by inverting the tube.
- Assemble a Filter Column with one of the provided collection tubes. Pipette the lysate into Filter Column and spin for 2 min at 20,000× g.
- Transfer only the clear supernatant from the flowthrough into an RNase-free microcentrifuge tube.
- Add an equal volume of 96–100% ethanol to the collected lysate. Vortex to mix.
- Binding to Column
- Assemble a Spin Column with one of the provided collection tubes.
- Apply up to 600 μL of the clarified lysate with ethanol onto the column and centrifuge for 1 min at ≥3500× g. Discard the flowthrough and reassemble the spin column with the collection tube.
- Depending on the lysate volume, repeat step 2b if necessary.
- Column Wash
- Apply 400 μL of Wash Solution A to the column and centrifuge for 1 min at 20,000× g.
- Discard the flowthrough and reassemble the spin column with its collection tube.
- Repeat steps 3a and 3b to wash the column a second time.
- Wash the column a third time by adding another 400 μL of Wash Solution A and centrifuging for 1 min.
- Discard the flowthrough and reassemble the spin column with its collection tube.
- Spin the column for 2 min at 20,000× g. Discard the collection tube.
- RNA Elution
- Place the column into a fresh Elution tube.
- Add 50 μL of Elution Solution A to the column.
- Centrifuge for 2 min at 200× g, followed by a 2 min spin at 20,000× g.
4.1.4. RNA Quality and Quantity Control
- Measure RNA concentration using a fluorometer (Qubit).
- Assess RNA integrity using a bioanalyzer.
- Evaluate purity ratios (A260/A280 and A260/A230) using a spectrophotometer (Nanodrop).
- Multiple sorbitol pre-washes were tested, but the results showed no significant improvements.
- The berry skin RNA extraction was performed on multiple samples to validate the method.
- The commercial kit-based extraction was extended to additional grapevine tissues (rachis, buds, and roots) for comparison.
- A graphical protocol is shown in Supplementary Figure S2.
4.2. qPCR Validation
4.3. Library Preparation and RNA Sequencing
4.4. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Tissue Type | # of Samples | Mean RNA Yield a | Mean A260/A280 b | Mean A260/A230 b | Mean RIN c | ||||
---|---|---|---|---|---|---|---|---|---|
Without Sorbitol | With Sorbitol | Without Sorbitol | With Sorbitol | Without Sorbitol | With Sorbitol | Without Sorbitol | With Sorbitol | ||
Rachises | 5 | 22.1 ± 11.5 | 6.4 ± 1.5 | 2.1 ± 0.1 | 2.1 ± 0.1 | 0.4 ± 0.2 | 0.1 ± 0 | 5.8 ± 1.9 | 0.9 ± 1.3 |
Buds | 5 | 28.5 ± 10.5 | 36.5 ± 5.6 | 2.1 ± 0 | 2.1 ± 0 | 0.6 ± 0.2 | 0.7 ± 0.1 | 6.4 ± 2.3 | 4.8 ± 1.1 |
Skins | 5 | 3.3 ± 3.4 | 20.8 ± 4 | 2.0 ± 0.3 | 2.1 ± 0.1 | 0.1 ± 0 | 1.2 ± 0.3 | 1.2 ± 1.6 | 7.2 ± 0.2 |
Roots | 5 | 16.1 ± 15 | 9.3 ± 7.6 | 1.8 ± 0.3 | 2.0 ± 0.1 | 0.4 ± 0.2 | 0.2 ± 0.1 | 4.9 ± 3 | 2.8 ± 2.8 |
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Prencipe, A.; Salerno, A.; D’Amico, M.; Marsico, A.D.; Ventura, M.; Velasco, R.; Cardone, M.F.; Bergamini, C.; Maggiolini, F.A.M. Optimized Protocol for High-Quality RNA Extraction from Grape Berry Skins Using Sorbitol Pre-Wash. Plants 2025, 14, 988. https://doi.org/10.3390/plants14070988
Prencipe A, Salerno A, D’Amico M, Marsico AD, Ventura M, Velasco R, Cardone MF, Bergamini C, Maggiolini FAM. Optimized Protocol for High-Quality RNA Extraction from Grape Berry Skins Using Sorbitol Pre-Wash. Plants. 2025; 14(7):988. https://doi.org/10.3390/plants14070988
Chicago/Turabian StylePrencipe, Annalisa, Antonella Salerno, Margherita D’Amico, Antonio Domenico Marsico, Mario Ventura, Riccardo Velasco, Maria Francesca Cardone, Carlo Bergamini, and Flavia Angela Maria Maggiolini. 2025. "Optimized Protocol for High-Quality RNA Extraction from Grape Berry Skins Using Sorbitol Pre-Wash" Plants 14, no. 7: 988. https://doi.org/10.3390/plants14070988
APA StylePrencipe, A., Salerno, A., D’Amico, M., Marsico, A. D., Ventura, M., Velasco, R., Cardone, M. F., Bergamini, C., & Maggiolini, F. A. M. (2025). Optimized Protocol for High-Quality RNA Extraction from Grape Berry Skins Using Sorbitol Pre-Wash. Plants, 14(7), 988. https://doi.org/10.3390/plants14070988