Expression of Pto-MIR475b and Its Target Genes Using Real-Time Quantitative PCR (RT-qPCR)

Total RNAs were extracted from eight different tissues: root, shoot apex, cambium, developing xylem, mature xylem, phloem, mature leaf, and young leaf. All samples were collected from a 1-year-old P. tomentosa clone, “LM50,” and stored in liquid nitrogen until RNA extraction. The total RNAs were extracted using the Plant Qiagen RNeasy kit (Qiagen China, Shanghai) following the manufacturer’s instructions and RNase-free DNase (Qiagen) was used to purify the total RNA. The total RNAs were reverse transcribed into cDNA with the Reverse Transcription System (Promega Corporation, Madison, WI, United States). RT-qPCR was performed on a 7500 Fast Real-Time PCR System using SYBR Premix Ex Taq according to the manufacturer’s protocol. The specific primers for each gene for qPCR (Supplementary Table S1) were designed by Primer Express 5.0 software (Applied Biosystems). The expression levels were normalized to poplar Actin (Accession number: {"type":"entrez-nucleotide","attrs":{"text":"EF145577","term_id":"118483655","term_text":"EF145577"}}EF145577). All reactions were performed in three technical and biological repetitions, and differential reactions across classes were tested by ANOVA (Analysis of Variance) as described by Song et al. (2013). The qPCR amplification program was as follows: initial denaturation at 94°C for 5 min; followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final melting curve of 70–95°C.