Approximately 50 mg of powdered fresh tissue was weighed out for RNA extraction using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions, with an on-column DNAse I digestion. RNA concentration was determined spectrophotometrically with Nanodrop at 260 nm (ND1000, Thermo Fisher Scientific, Waltham, MA, USA) with a desired ratio of 260/280 ∼ 2.0 and 260/230 ∼ 2.0-2.3. Additionally, the quality of selected RNA samples was checked using the bioanalyzer (2100 bioanalyzer, Agilent Technologies). A RIN value of ≥ 7.3 was accepted for further usage. The cDNA was synthesized with the SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and oligo (dT)12−18 primers as described by the manufacturer using 250 ng total RNA. Primers for target and reference genes were designed using sequences available at Phytozome or NCBI (National Center for Biotechnology Information; Table S4). The primer amplification efficiencies were determined with cDNA dilution analysis. Detailed information about primer sequences and efficiencies can be found in Table S3. The stability of selected reference genes (actin 7, ubiquitin-conjugating enzyme E2 A and elongation factor 1-alpha) was checked (M value <0.5; coefficient variance <0.25). The RT-qPCR experiments were performed in triplicates using 3 μL diluted cDNA (1:10), 5 μL 2× SensiMix SYBR Low-ROX (Bioline, Luckenwalde, Germany) and 2 μL of 2 μM primer. Experiments were conducted with a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the following thermal cycling conditions: 95°C for 10 min, 39 cycles of 95°C for 15 s, 58°C for 15 s followed by 72°C for 30 s and a subsequent melting curve analysis. For the analysis of CCD4 and OR family genes, an adjusted annealing temperature of 60°C was used. Data were evaluated using the ΔΔCq method according to Vandesompele et al. (2002) (link); Pfaffl (2004) with the geometric mean of the three reference genes. The expression of genes of interest were calculated as n-fold changes relative to gene expression in the lettuce samples grown without polytunnels.
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