For this purpose, rosette leaf tissue was harvested from 4-week-old T4 generation wild-type and CBF transgenic Arabidopsis plants between 10 and 11 am (i.e. 3?C4?h after starting the light period), flash frozen in liquid PD 98059 N2 and stored at ?80???C until RNA extraction. The whole leaves were ground to a fine powder in liquid nitrogen. RNA was extracted with an RNeasy Plant Mini-Kit (Qiagen, Mississauga, ON, Canada), subsequently DNase treated with RNase free DNase (Ambion, Invitrogen, Carlsbad, CA, USA) and used for cDNA synthesis with qScript cDNA Super mix (Quanta Biosciences, VWR, Canada) according to the instructions and components given by the manufacturers. One microgram DNA-free RNA was used as a template for cDNA synthesis. Real-time PCR was performed in 20??L of reaction mixture using PerfecTa SYBR Green Supermix (Quanta Biosciences) in an Applied Biosystems 7300 thermocycler. The reaction mixture containing 50?ng cDNA and 0.2??m of each primer was RAD001 subjected to one cycle of 95???C for 10?min, followed by 40 cycles of 95???C for 15?s and 60???C for 1?min. The following gene-specific primers were used: VrCBF1-H565 (5?? AAGAGGTCAGAGAAGGTTGGAGATG 3??), VrCBF1-C684 (5?? AGCAGGTGGAGTAAGGAGCAAAC 3??), VrCBF4-H19 (5?? ACCCTCACCCGCTCGTATG 3??), VrCBF4-C159 (5?? CCGCGTCTCCCGAAACTT 3??), AtCor15a-Hq (5?? TAAAGGTGACGGCAACATCC) and AtCor15a-Cq (5?? TCGCTTTCTCACCATCTGCT 3??), AtRD29A-Hq (5?? GAACACTCCGGTCTCTCTGC 3??) and AtRD29A-Cq (5?? CAATCTCCGGTACTCCTCCA 3??), AtCor6.6-Hq (5?? TGGGAGTCATAATTGATTCTCGTA 3??) and AtCor6.6-Cq (5?? AAGTTCCAAACGTAGTACATCTAAAGG 3??), AtCor47-Hq (5?? AGCGATGAAGAAGGTGAGGA 3??) and AtCor47-Cq (5?? ACACTGGTACCGGGATGGTA 3??), AtA2ox7-Hq (5?? GCCATCTAACTAGTGGTGAGGAGGT 3??) and AtGA2ox7-Cq (5?? TCCCCACTCTTTCGCAGCT 3??), AtGA2ox3-Hq (5?? GGCACACCCCTGCAATTTT 3??) and AtGA2ox3-Cq (5?? CCAGAAATTTGCTCGACATTCTC 3??), AtRGL3-Hq (5?? ATGGATACAGAGTGGAGGAGAACG 3??) and AtRGL3-Cq (5?? GATGCAGCGATTAGAGGTTTCG 3??), AtFLC-Hq (5?? GAGAATAATCATCATGTGGGAGC buy Sirolimus 3??) and AtFLC-Cq (5?? CAACCGCCGATTTAAGGTGG 3??), AtICE1-Hq (5?? TCCTAAAGGCCAGCAAGCTA 3??) and AtICE1-Cq (5?? CTCTTGTCCTTCTTGGCATTG 3??), AteIF1alphaH769 (5?? CTTCGTCTTCCACTTCAGGATGT 3??) and AteIF1alphaC889 (5?? TCAACCCTGTGGGAGCAAAG 3??). The relative quantification of mRNA levels was determined by normalizing the PCR threshold cycle (CT) number of each gene with that of the EF1?? reference gene. The expression level of each gene in the wild-type control was set to 1. For VrCBF1 and VrCBF4, the amplification on 10?pg plasmid DNA containing VrCBF1 and VrCBF4 coding sequence, respectively was set to 1. Data were analysed by one-way analysis of variance. Statistical differences among the means were determined by the Tukey?CKramer HSD tests (P?<?0.05) using JMP (version 8.0 SAS Institute, Cary, NC, USA) statistical software.