Opticon monitor analysis software 3.1 tool

Real-time quantitative PCR was performed on a DNA Engine Opticon 2 machine (MJ Research, Bio-Rad, Hercules, CA, USA) using the Light Cycler-Fast Star DNA master SYBR Green I kit (Roche, Basel, Switzerland). The PtoXET16A-specific and internal control (Actin) primer pairs were designed using Primer Express 3.0 software (Applied Biosystems, Life Technologies, New York, NY, USA). The PCR program included an initial denaturation at 94 °C for 5 min, and 40 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and a final melt-curve of 70–95 °C. The specificity of the amplified fragments was checked by melting curve. All reactions were carried out in triplicate, and the data were analyzed using the Opticon Monitor Analysis Software 3.1 tool (MJ Research, Bio-Rad, Hercules, CA, USA).

A 7500 Fast Real-time PCR System (Applied Biosystems) was used for RT-qPCR analyses with SYBR Premix Ex Taq (TaKaRa). The cDNA template for reactions was reverse-transcribed using the total RNA extracted from leaves subjected to the salt stress treatments. The real-time data generated were analyzed using the Opticon Monitor Analysis Software 3.1 tool (Bio-Rad), and quantities of mRNA were standardized to the transcript levels of poplar ACTINII-like (accession number EF145577) and 18S ribosomal RNA (18S rRNA), which were used as internal controls, and then calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The PCR program was as described by Zhang et al. For all reactions, three technical and biological repetitions were performed, and the specificity of the amplified fragments was checked using generated melting curves.