2.7. qRT-PCR Validation and Analysis

qRT-PCR was conducted to validate the expression profiles of auxin early response genes derived from RNA sequencing. Twelve representative genes including two ARF, six Aux/IAA, two GH3, and two SAUR genes were selected for qRT-PCR validation. Specific primers were designed using the National Center for Biotechnology Information (NCBI) Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) and synthesized commercially (Sangon Biotech). cDNA was synthesized from RNA samples of NEC and EC of CCRI12 and CCRI24 using a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co. Ltd, Nanjing, China) according to the manufacturer’s instructions. The obtained cDNA products were diluted five times prior to amplification. qPCR was conducted with a 20 μL reaction system containing 1 μL cDNA templates, 10 μL 2 × TransStart Top Green qPCR Super Mix (TransGen Biotech Co. Ltd, Beijing, China), 0.5 μL of each 10 μM forward and reverse primers, and 8 μL ddH₂O. All qPCR reactions were performed in triplicate on a Roche Light Cycler 480 instrument. A thermal cycling program was performed with pre-incubation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 10 s and extension at 72 °C for 10 s. Relative quantitation of gene expression was computed using the online tool A shiny for the analysis of real-time PCR data (https://ihope.shinyapps.io/qRT-PCR-Pipeline/) with GhHis3 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"AF024716","term_id":"2558943","term_text":"AF024716"}}AF024716) was used as the internal reference.