First strand cdna synthesis kit

Total RNA was isolated from cells with GenElute mammalian total RNA miniprep kit (Sigma-Aldrich). Reverse transcription (RT) was performed with 1 μg RNA using the first-strand cDNA synthesis kit (Quanta Bioscience, GE Healthcare). qRT-PCR was performed with cDNA diluted 1:25 and the iTaq Universal SYBR Green Supermix reaction kit (Biorad) in combination with the Applied Biosystems 7500 thermal cycler (Applied Biosystems). EGFR relative mRNA expression was determined using the ΔΔCt data analysis method [45 (link)] using the EGFR forward 5′-tgcgtctcttgccggaat-3′ and reverse 5′-ggctcaccctccagaaggtt-3′ primers [46 (link)]. The hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene was used as housekeeping gene and its expression was assessed with the HPRT1 forward 5′–gtaattggtggagatgatctctcaact-3′ and reverse 5′-tgttttgccagtgtcaattatatcttc-3′ primers [47 (link)].

All the samples were harvested from 3:00 pm and ground to a fine powder in liquid nitrogen. Total RNA was isolated using Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Total RNA (1 μg) was used to synthesize cDNA with the first Strand cDNA Synthesis Kit (Quanta Biosciences, USA). 15 ng cDNA was used for qPCR analysis. qPCR was completed in a 20 μL solution containing 5 μM specific primers and 2 × SYBR Green/Fluorescein qPCR Master Mix (thermo scientific), and program was as follows: 95 °C for 4 min; 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The melting curve analysis was completed by increasing the temperature from 60 to 95 °C at a rate of 0.05 °C s⁻¹. Relative expression levels of genes related with fructan metabolism in this manuscript were calculated using the comparative Ct method^{8 (link),31 (link)}. Gene expression levels normalized against the values of the housekeeping gene Ubiquitin10. All the primers used for qPCR were listed in Supplementary Table 2.