Biorad iq5 pcr thermal cycler

Total RNA was isolated from cucumber fruits using Trizol reagent (Tiandz, Beijing, Inc.) and treated with RNase-free DNase (Tiandz, Beijing, Inc) to remove genomic DNA. Reverse transcription was performed using approximately 1 μg of purified RNA as template with oligo(dT)₁₈ and a Revert Aid First Strand cDNA Synthesis Kit (Fermentas) following the manufacturer’s instructions. CsHPL and CsLOX gene-specific primers for RT-qPCR were designed based on the CsHPL sequence (Accession No. AF229811) and CsLOX sequence (Accession No. KC429651), respectively. Quantitative PCR was performed using SYBR Green in a BioRad IQ5 PCR thermal cycler (Bio-Rad Co., USA). Real-time PCR conditions consisted of 95°C for 1 min, followed by followed by 40 cycles of 95°C for 15 s, 57°C for 30 s, and 72°C for 20 s. Actin cDNA was used as an internal control. The relative expression levels of LOX, HPL and other genes were calculated by the ΔΔCT method. PCR primers were designed using primer Premier 5.0 as follows; for CsLOX, CsLOX-RT F: 5′-GGA GAT GGT ACT GGA GAG CG-3′ and CsLOX-RT R: 5′-CAC GAC GAG GGT AAG GGA A-3′; for CsHPL, CsHPL-RT F: 5′-CTC CTT TCT CGC TTC TCA CC-3′ and CsHPL-RT R: 5′-C TCA AAC GAC ACG GCA TCA CT-3′.

Total RNA isolation was performed according to Wan et al. [46 (link)]. Gene-specific primers for RT–qPCR were designed using Primer 5, and the primer sequence is given in Supplementary Data Table S7. Gene expression analysis of CsLOXs was performed using SYBR Green in a BioRad IQ5 PCR thermal cycler (Bio-Rad, USA). The associations between CsLOX gene expression and alcohol content were investigated using Graphpad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA).