Quantitative Gene Expression Analysis in Poplar

Total RNA was extracted using a Plant RNA EASYspin Plus Kit (Aidlab, Beijing, China) and first-strand cDNA synthesis was performed using approximately 2 μg of RNA using the FastQuant RT Kit (with gDNase) (TIANGEN, Beijing, China) following the manufacturer’s protocols. qRT-PCR was conducted on the ABI StepOnePlus Real-Time PCR System (ABI, Foster City, CA, USA) based on the SYBR Green II method. Twenty microliters of cDNA was diluted 1:10 with nuclease-free water. Each reaction contained 10 µL of SYBR Green qPCR Mix (Aidlab), 0.2 µL of ROX Reference Dye (Aidlab), 1 µL of cDNA (corresponding to 10 ng of total RNA), 7.8 µL of nuclease-free water, and 0.25 µM each primer. The thermal profile for qRT-PCR: 3 min at 94 °C, 40 cycles of 10 s at 94 °C, 20 s at 60 °C, and 72 °C for 30 s. Primers were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) [69 (link)]. A list of primers used was given (Table S4). Each experiment was performed in three biological replicates. The poplar housekeeping gene, UBQ, was used as an internal control. Relative expression was calculated by the 2^−ΔΔCt method [70 (link)]. A Student’s t-test was used to generate every p-value for statistical analyses, and R project was used to identify significant variance (R version 3.5.1, one sample and two sample t-test: * p < 0.05; ** p < 0.01).