5.3. Transcript Analysis

Frozen rosette leaves were ground to a fine powder under liquid N₂ using a sterile mortar and pestle. Total RNA was extracted from 30 mg fresh weight of frozen tissue and cDNA synthesis was performed as previously described [53,54]. Up to 5 µl of diluted cDNA was used in qPCR reactions contained in a total volume of 20 µl. The gene-specific primers for reference genes Actin 2 (ACT2) and Transcription Elongation Factor (TEF) and for CBF1-3, COR78, COR15a, and ERD10 from Arabidopsis were previously described [55]. All other primer pairs were designed using the Primer 3 program and primer sequences used for qPCR are listed in Table S2. qPCR was performed as previously described [54] and relative transcript abundance was measured by normalizing the expression level of each gene of interest using the expression of reference gene ACT2. ACT2 and TEF showed the same expression pattern, but % PCR efficiency of ACT2 was greater than TEF (97 vs. 94% respectively); therefore, ACT2 data were used for normalization. The presence of a single PCR product was further verified by dissociation analysis. Three independent experiments were performed with three to four plants per treatment. Significant differences were determined using a one-way or two-way ANOVA procedure in combination with Tukey HSD (StatView 5.0, Mountain View, CA, USA).