Stratagene mx3005p real time pcr instrument

We used qRT-PCR to analyze the expression profiles of MSTRG.20413.1, ptc-miR396e-5p, and the corresponding target gene (GRF9) within 24 h of blue and white light irradiation. Primers were designed using Primer 6.0 software, as shown in Table S3. RNA was reverse-transcribed into cDNA using the miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN Biotech, Co., Ltd., Beijing, China) to generate a fluorescent quantification template for ptc-miR396e-5p. Amplification was performed using the miRcute Plus miRNA qPCR (SYBR Green) Kit (TIANGEN Biotech, Co., Ltd., Beijing, China). The reaction system included 10 μL of 2×miRcute Plus miRNA PreMix (SYBR&ROX), 0.4 μL each of forward and reverse primers, 1 μL of miRNA first-strand cDNA, and 8.2 μL of ddH₂O. The amplification program consisted of an initial denaturation at 95 °C for 15 min, followed by 43 cycles of denaturation at 94 °C for 20 s, and annealing at 60 °C for 34 s. U6 was selected as the reference gene. The reaction system and program for GRF9 were set up and performed in the same manner as described in Section 4.6 for the fluorescent quantification of target genes. The Stratagene Mx3005P Real-Time PCR instrument (Agilent Technologies) was used for qRT-PCR. The relative expression level of the gene was calculated using the 2^−ΔΔCT method.

In early July, three biological replicate samples of fully expanded young leaves from all transgenic lines and non-transgenic Populus × euramericana ‘Neva’ (CK) were collected from the field, frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted and reverse transcribed to produced first-strand cDNA using RNA extraction and reverse transcription kits (SENO, Zhangjiakou, Hebei, China) according to the manufacturer’s instructions. The FQ-PCR primers 176# and 1291# (amplified target fragments were 176 and 203 bp, respectively; Supplementary Table 1) were designed according to the full Cry1Ac and Cry3A nucleotide sequences using Primer Premier 6.0 (Premier Biosoft, Canada) software. Absolute FQ-PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) and an Agilent Technologies Stratagene Mx3005P Real-Time PCR instrument (Agilent, United States) to determine the transcriptional expression levels of the exogenous genes. FQ-PCR was performed according to Liu et al. (2016) (link).