Plant materials
Fully extended leaves and ripe fruits were harvested from 3-month-old strawberry plants (Fragaria × ananassa Duch. cv Fertona) after transplanting. Both tissues were immediately shock-frozen in liquid nitrogen and then stored in -80°C for subsequent extraction of RNA and semi-quantitative RT-PCR.
Buffers
Lysis buffer 1 [13]: 100 mM Tris-HCl, pH 8, 1.4% NaCl, 0.5% (v/v) Triton X-100 and 3% (w/v) CTAB (cetyl trimethyl-ammonium bromide). 2% (w/v) poly-vinyl pyrrolidone (PVP), and 5% (v/v) 2-mercaptoethanol (β-ME) were added just before use. Prewashing buffer (only in case of fruits) [14]: 100 mM Tris-HCl, pH 8, 350 mM sorbitol, 5 mM EDTA, pH 8.0 (the buffer should be stored at + 4 °C), in addition 3% (v/v) β-ME was added immediately before use.
Reagents
Trizol (Ambion, Invitrogene, USA), chloroform:isoamylalcohol (24:1), isopropanol, 75% ethanol, absolute ethanol, and DNase-RNase free water, or double-distilled water.
Kits
Direct-zol RNA miniprep (Zymo Research, USA).
RNA isolation via the double-lysis protocol:
All centrifugation steps must be performed under cooling (+ 4 °C)
Note: while the dissolution of the sediments requires several steps of short vortexing during preparation, never vortex the purified RNA, especially while dissolving it in water.
1. Weigh in maximally 0.03 g of leaf tissue or nearly 0.5 g of fruit tissue.
2. Grind the tissue in liquid nitrogen to fine powder using mortar and pestle.
For fruits only, add 1 ml of prewashing sorbitol buffer (ice-cold) supplied with 30 μl β-ME for each sample, vortex thoroughly for 30 s, then spin down for 5 min at 3700×g on 4 °C. Completely discard the supernatant including any floating tissues. Repeat this step once more and then go to step 3.
For leaves, go directly to step 3.
3. Add 1 ml of the lysis buffer (buffer 1) containing freshly added 50 μl of β-ME and 20 mg PVP. Resuspend the sediment by pipetting several times with cut tips till the tissue is resuspended completely, then vortex 30 s.
4. Incubate 10 min at 65 °C, invert the tubes gently a couple of times every 2 min (do not vortex during heat incubation) and centrifuge 5 min at 17,600×g on 4 °C.
5. Transfer 700 μl of the supernatant into a separate tube and add an equal volume of chloroform:isoamylalcohol (24:1). Vortex 10 s, then let settle at room temperature for 5 min. Spin down 15 min at 21,000×g on 4 °C.
6. Take the 400 μl supernatant and add 800 μl Trizol (ice cold), vortex 30 s, then let it at room temperature for 5 min.
7. Add 200 μl of chloroform, vortex 10 s, and incubate at room temperature for 5 min.
8. Spin down for 15 min at 21,000×g on 4 °C, and then transfer the upper 750 μl from the upper phase into a separate tube, where 500 μl isopropanol are added, and the mixture is then mixed by pipetting several times. Incubate the mixture at room temperature for 10–15 min, then transfer to – 20 °C for 10–15min.
9. Spin down for 15 min at 17,600×g on 4 °C, to obtain a visible precipitate and discard the supernatant.
10. Add 1 ml of 75% cold ethanol, then vortex 10 s. Re-spin for 3 min at 17,600×g and 4 °C. Discard the supernatant.
11. Repeat step 10.
12. Add 1 ml of absolute ethanol, vortex 10 s, then spin down for 5 min at 17,600×g on 4 °C.
13. Carefully discard the supernatant completely and let the precipitates to dry for 5 min only at room temperature. Do not lose the precipitate as at that stage it tends to be loose.
14. Add 30–50 μl of DNase-RNase free water (do not pipette or vortex) and keep in room temperature for 15 min then dissolve the white sediments carefully by gentle pipetting for few times. Note: never vortex at that stage, since it will disrupt RNA integrity.
RNA isolation from strawberry petals and roots by double-lysis method
The protocol applied with leaves was also used to isolate RNA from petals (0.3 g) and roots (0.1 g) of strawberry plants, just omitting the prewashing step.
Other used protocols:
- 1)
Trizol (Ambion, USA) according to the manufacturer instructions.
- 2)
Direct-zolTM RNA miniprep according to the manufacturer instructions.
RNA quantification and purity level
The RNA was quantified spectroscopically (Nanodrop® Thermo Scientific, USA) at 260 nm. The level of purity was inferred from the ratios of A260/A280 for protein contamination, and A260/A230 for determining the contamination with polysaccharides and polyphenolics.
DNase digestion
The isolated RNA of all samples was further digested with DNase using the product DNase I, RNase-free (Thermo Scientific, USA) according to the manufacturer instructions. The obtained RNA was directly used in subsequent cDNA synthesis reactions. It is recommended to not add more than 1 μl of DNase for more than 1 μg of isolated RNA, since this might affect RNA integrity.
Reverse transcription-polymerase chain reaction (RT-PCR)
The cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit (Thermo Scientific, USA). As template, 280 ng of RNA for at least three samples of leaves and fruits were added to 1 μl of oligo (dT)18 primers, mixed well, and heated at 65 °C for 5 min, before keeping the sample on ice for at least 2 min. Subsequently, the following mixture is added: 5× reaction buffer (1 μl), RiboLock RNase Inhibitor, 20 U/μL (1 μl), 10 mM dNTP Mix (2 μl), and RevertAid M-MuLV Reverse Transcriptase, 200 U/μl (1 μl). The whole mixture was incubated at 42 °C for 60 min and at 70 °C for 5 min to deactivate the reverse transcriptase and terminate the reaction. The mixture was then cooled on ice for at least 5 min and then diluted tenfold with 10 DEPC-treated water. Aliquots of 3 μl were used to amplify FaGAPDH2 (AF421493.1) encoding glyceraldhyde-3-phophate dehydrogenase using the forward primer 5′-CTTGAGAAGAAGGCCACCTATG-3′, and reverse primer 5′-CTTCGGTGTAACCCAAGATACC-3′. The product size should be 91 bp in case of purified RNA with no DNA contamination, but 200 bp with DNA template [15]. The PCR was run using the ready-to-use reaction mixture Cosmo PCR red master mix (Willowfort, UK) according to the manufacturer protocol using initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s, and synthesis at 72 °C for 1 min. The final extension was conducted at 72 °C for 5 min. The reaction was stopped by pausing the reaction at 4 °C.
Visualization of RNA or PCR products
The isolated RNA and the PCR amplicons were separated on an 1.5% agarose gel at 5 V/cm for 30 min and visualized with ethidium bromide along with a 100-bp size marker (Thermo Scientific, USA).