Sybr green premix ex taq 2

Total RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China), reverse transcription was performed using PrimeScrip™ RT Master Mix (TaKaRa), and cDNA amplification was performed using SYBR Green Premix Ex Taq™ II (Takara) according to the manufacturer’s instructions. miRNA was extracted using an miRNeasy Mini Kit (Qiagen, Duesseldorf, Germany), reverse transcription was performed using an miScript II RT Kit (Qiagen), and cDNA amplification was performed using an miScript SYBR Green PCR Kit (Qiagen) according to the manufacturer’s instructions. The primers are listed in Additional file 1: Table S1.

Total RNA was extracted from Cd-treated and WT poplar samples using the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing), after which 800 ng total RNA in 20 μL was used to synthesize cDNA. The qRT-PCR analysis was performed using SYBR Green Premix Ex Taq II (TAKARA, Japan). The primer sequence refers to He et al. (2015) (link) and Ma et al. (2014) (link). Information regarding the qRT-PCR primers is provided in Table S1. The qRT-PCR reaction mixture was prepared in a volume of 20 μL, with 10 μL of SYBR green master mix, cDNA template (2 μL), ddH₂O (6.8 μL), ROX Reference Dye 0.4μL and 0.4 μL of each of the primer. The qRT-PCR conditions were set as follows; 95°C for 30s, 40 cycles of 95°C for 5 s, and 60°C for 30 s, followed by melting of PCR products step: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. Each sample was analyzed using three technical replicates to ensure the results were reliable. Gene expression levels were calculated according to the 2^−ΔΔCt method, with TUBU selected as the reference gene. Relative gene expression levels were visualized in a heatmap on the basis of Z-score transformed values.

100 mg tissue or 1 × 10⁶ cells were collected, and cells were lysed with TRIzol reagenet (Invitrogene, Carlsbad, CA, US) and total RNA was extracted. After modulating the concentration of RNA to 300–500 ng/μL, RNA was reversely transcribed by reverse transcription kit (Invitrogene, Carlsbad, CA, US) to produce cDNA. Next, RT-PCR was performed using SYBR Green Premix Ex Taq II (TaKaRa, Dalian, China) in accordance with the manufacturer’s protocol. GAPDH was regarded as the standard internal reference of MCM3AP-AS1 and WNT5A. Besides, U6 was used as the standard internal parameter of miR-876-5p. Relative quantitative method 2(^−ΔΔCt) was utilized to calculate the results. The experiment was repeated three times independently. The specific primer sequence information was listed in Table 1.Table 1

Primer sequence of qRT-PCR

Name	Primer sequences (5′ to 3′)
MCM3AP-AS1	Forward: 5′-GCTGCTAATGGCAACACTGA-3′
	Reverse: 5′-AGGTGCTGTCTGGTGGAGAT-3′
GAPDH	Forward: 5′-CAGGAGGCATTGCTGATGAT-3′
	Reverse: 5′-GAAGGCTGGGGCTCATTT-3′
miR-876-5p	Forward: 5′-TGAAGTGCTGTGGATTTCTTTGTG-3′
	Reverse: 5′-TGAATTACTTTGTAAACCACCACCA-3′
WNT5A	Forward: 5′-CGCCCAGGTTGTAATTGAAG-3′
	Reverse: 5′-GCA TGTGGTCCTGATACAAGT-3′
U6	Forward: 5′-GTGGACCGCACAAGCTCGCT-3′
	Reverse: 5′-TTGTTGAACGGCACTGTGTATAGCA-3′

RT-qPCR was performed on a 7,500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, United States) using the SYBR Green Premix Ex Taq II (TaKaRa). All primer pairs for the candidate genes were designed by an online tool provided by Integrated DNA Technologies⁶, as shown in Supplementary Data 1. Poplar 18S rRNA was used as an internal control for gene expression measurements for target genes. The Mir-X^TM miRNA qRT-PCR SYBR Kit (Clontech, Mountain View, CA) was used. The relative expression level of each miRNA was measured and standardized to 5.8S rRNA. The relative expression of miRNAs and target genes was calculated using the 2^–△△Ct method.

Real‐time polymerase chain reaction (RT‐PCR) was carried out to detect mRNA expression. Total RNA was extracted from NRCs using TRIzol reagent (Invitrogen) according to manufacturer's instructions. The concentration of RNA was quantified with a NanoDrop spectrophotometer (Thermo Fisher), and the RNA was transcribed into cDNA using a PrimeScript cDNA Synthesis Kit (Takara, Japan). For real‐time quantitative PCR, SYBR Green Premix Ex Taq II (Takara, Japan) was used on 480 II PCR Sequence Detection System (Roche). Relative mRNA expression level was normalized to the expression level of GAPDH and expressed relative to that of the control group. Primer sequences used are listed in Table 1 (Supplementary material).