Mx3000p

Cementoblasts cultured in 6-well plates were incubated with strontium at 12.5 μM for 3 days. Total RNA was extracted using RNAiso Plus (Takara Co., Japan) according to the manufactures' instructions. Then RNA was analyzed for quality and quantity by measuring the A260/A280 ratio with ultraviolet spectrophotometry. 2 μg of total RNA was used in following reverse transcription reaction by employing PrimeScript RT reagent kit (Takara Co., Japan). Then each sample was analyzed by quantitative real-time PCR (qPCR) (Stratagene MX3000P, Japan) in the SYBR Premix Ex TapII (Takara Co., Japan), setting the cycles as follows: 10 s/95°C PCR initial activation step, 40 cycles of denaturation for 20 s/95°C, and annealing step for 20 s/60°C. Formula 2^−(ΔΔCT) was used to determine the change in mRNA levels, where ΔCT is the value from the threshold cycle (CT) of the treated sample subtracted from the CT value of untreated or zero time-point control samples. Normalization to GAPDH mRNA was performed to decide the relative amount of mRNA in the sample. Primers used were listed in Table 1.

The total RNA of leaves was extracted by RNeasy plant mini kit (QIAGEN) following the manufacturer's instruction. Gene expression level analysis was performed by quantitative real-time PCR using a model real-time PCR system (Agilent Mx3000P) with SYBR Premix Ex Taq™ (Takara). Specific primers used in quantitative real-time (qRT)-PCR were listed in Table S3. Relative gene expression levels were normalized to reference gene NtActin. Real-time PCR data were calculated with the comparative Ct method. Three biological replicates were performed for each amplification and each PCR was performed in triplicates independently.

Total RNA was extracted from approximately 0.1 g leaves with RNeasy plant mini kit (QIAGEN). Synthesis of the first strand of cDNA was performed using 2 μg of total RNA primed with oligo (dT)₁₈ primers by PrimeScript™ RT Master Mix (for Real-time) kit. Real-time PCR was performed with a model real-time PCR system (Agilent Mx3000P) using SYBR Premix Ex Taq TM (Takara) according to the manufacturer's guidance. To normalize the results, NtActin was amplified for each sample as a control to calculate ΔCt (ΔCt = Control Ct − Target Ct) values, and gene-specific primers involved in real-time PCR were listed in Table S5. Thermal cycling conditions included an initial incubation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 58 °C for 35 s, and 72 °C for 15 s. All the samples were run in triplicate, and the standard deviation was calculated.