Primescript rt reagent kit

Sixteen unigenes (10 unigenes for original transcriptome verification and six unigenes for commercial insecticide verification) were selected and validated by quantitative real-time PCR (qRT-PCR) on a CFX-96 Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA) with specific primers designed by Primer Premier 5.0 (Table S1). The cDNA template used for RT-qPCR was reverse-transcribed from qualified total RNA using the Prime Script RT Reagent Kit (Bio-Rad, Berkeley, CA, USA). A 20 µL PCR reaction volume contained 10 µL of Bio-Rad iTaq™ Universal SYBR^® Green Super mix (2 ×), 1 µL of each gene-specific primer (10 µM), 2 µL of each cDNA template and 6 µL of ddH₂O. Actin was used as a reference gene and the 2^−ΔΔCt method was employed to estimate relative changes in gene expression (Livak & Schmittgen, 2001 (link); Chang et al., 2017a (link)). Each treatment included four replicates, and each sample was assessed in triplicate. Significant differences among treatments were detected by one-way ANOVA, followed by Tukey’s multiple comparison and analysis with SPSS v. 16.0 (SPSS, Chicago, IL, USA); significant differences were determined at P < 0.05.

Cells were washed by PBS and 1ml/ 3.5cm diameter well, tissues were crushed by pestle and mortal, then add 2ml/ 1g Trizol (Introvergen). Total RNA was extracted from tissues or cells, according to the manufacturer's instructions. Reverse-transcription PCR (RT-PCR) was carried out with the Prime-Script RT Reagent Kit (Bio-Rad). Expression levels of the genes were determined by real-time quantitative PCR and normalized against an endogenous control GAPDH using SYBR supermix (Bio-Rad). Data were analyzed using a ΔΔCq approach.

qRT‒PCR was used to quantify the mRNA expression of adhesion-related and osteogenic differentiation-related genes in the cells of each group. After culture for 2 or 6 h, total RNA was extracted from the cells according to the manufacturer’s protocol (Takara, USA). Nanodrop plates (Tecan Infinite M200, USA) were used to measure the concentration and purity of RNA. The RNA content in each group was adjusted to 1 μg, and reverse transcription was performed using the PrimeScript RT Reagent Kit (Bio-Rad, USA). qRT‒PCR was performed with a CFX96 fluorescence quantitative PCR machine (Bio-Rad, USA), and the key adhesion-related genes integrin β1, vinculin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were assessed. β-actin was used as the reference gene to standardize the expression levels of other genes. The standard ΔΔCt (threshold cycle) method was used to analyze the experimental data. To detect osteogenic differentiation, the cells were cultured for 5 d following the method outlined above. qRT‒PCR was completed using a SYBR premix EX Taq PCR kit (Takara, Japan) to evaluate the mRNA levels of alkaline phosphatase (ALP), run-related transcription Factor 2 (Runx2), and osteopontin (OPN). The mRNA levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).