Quant one step qrt pcr kit sybr green

MiR-30c, miR-30d and miR-30e-3p were chosen as the candidate miRNAs for microarray data validation. U6 RNA was chosen as an internal control. The 20 μL reverse transcriptase reactions contained 1 μg of RNA and were completed using Mircute enhanced miRNA cDNA first strand synthesis Kit (TIANGEN, China). Real-time PCR was performed using Mircute enhanced miRNA fluorescence quantitative detection kit (TIANGEN, China). The genes of Ubadc1, Ldhb, and Cabc1 were selected to validate the microarray results by Quant one step qRT-PCR Kit (SYBR Green) (TIANGEN, China). β-actin was chosen as an internal control. The sequences of the PCR primers (synthesized by Sangon Biotech Co, China) were shown in Table S1.

The mRNA was isolated with the RNAprep Pure Plant Kit (Tiangen, China) and stored at −80 °C for quantitative real-time PCR (RT-qPCR). Twenty genes involved in fructose and mannose metabolism and flavonoid biosynthesis were randomly selected for RT-qPCR. All reactions were carried out in eight strip RT-PCR tubes in the Light Cycler 480 II System (Roche, USA) using the Quant One Step qRT-PCR Kit (SYBR Green) (Tiangen, Beijing, China). The PCR programs were as follows: 50 °C for 30 min, 95 °C for 2 min, and 40 rounds of 94 °C for 20 s, 55 °C for 20 s, and 68 °C for 20 s. The expression of all genes were normalized to 18S rRNA (Cluster-168003.101068) and calculated by the 2^−ΔΔCt method. Each gene has three biological replicates. All primers in the experience were listed in Supplementary Table S7.