Dna engine opticon 2 machine

For quantitative PCR analysis, the TaKaRa ExTaq R PCR Kit, SYBR green dye (TaKaRa, Dalian, China) and a DNA Engine Opticon 2 machine (MJ Research) were used. Gene-specific primers were designed to target the 3′ UTR of each gene (Supplementary Table S1). A melting curve was used to check the specificity of each amplified fragment. For all reactions, triplicate technical and biological repetitions of each individual were performed; the PCR was performed according to Song et al. (2014) (link). After amplification, the PCR products were sequenced to check the specificity of the primer sets. Relative expression levels of candidate genes were standardized to the transcript levels for PsiACTIN, which shows stable expression under abiotic stress calculated by the 2–^ΔΔCt method.

qRT-PCR was performed as described [21 (link)], using the TaKaRa ExTaq R PCR Kit, SYBR green dye (TaKaRa, Dalian, China) and a DNA Engine Opticon 2 machine (MJ Research, Waltham, MA). Fifteen genes including cellulose and lignin biosynthesis genes (Pt-CESA2.1, Pt-ATH.2, Pt-GLAC90.1, Pt-PRX1.8, Pt-PAL1.2 and Pt-PAL1.3) were validated, and the primers are shown in Additional file 1. The efficiency of the primers was calculated by performing real-time PCR on several dilutions of first-strand cDNAs. Efficiencies of the different primer sets were similar. The specificity of each primer set was checked by sequencing PCR products. The reactions were carried out in a 20 μl volume containing 2 μl of diluted cDNA, 200 nM of each primer, and PCR Master Mix with the following conditions: 95°C for 30 s, and 45 cycles of 95°C for 5 s, 58°C for 15 s, and 72°C for 20 s. Then, a thermal denaturing cycle of 95°C for 15 s and 60°C for 1 min was applied to determine the dissociation curves, which were used to verify the specificity of PCR amplifications. All reactions were run in triplicate for each sample. Relative expression levels of candidate genes were calculated by the 2^−ΔCt method. The results obtained for the different tissues were analyzed and standardized to the mRNA levels of poplar ACTINII-like (Accession number: EF145577), which shows stable expression.