Primer design and amplification efficiency analysis for RT-qPCR

Gene specific primers for RT-qPCR were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers are detailed in Table 1. The assay was performed using Rotor Gene Corbett 6000 quantitative real-time PCR system (Qiagen). The size of the PCR products was between 95–145 bp and annealing temperature was optimized to 60 °C. cDNA was amplified using the PowerUp SYBR Green Master mix (ThermoFisher Scientific) following manufacturer instructions with a final reaction volume of 20 μl in each well containing 4 μl cDNA, 10 μl SYBR Green Master mix, 1 μl (10 μM) of each sense and anti-sense primer and 4 μl PCR-grade water. The PCR reaction was carried out with a hold temperature of 50 °C for 2 mins and 95 °C for 2 mins followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 1 min. To increase the accuracy and produce reliable results, all qPCR analyses were conducted with three replicates. For each primer pair, amplifications included a no-template control to ensure the absence of other contamination or primer dimer. Melting curve analysis was performed at the end of each PCR to verify primer specificity. Amplicons from each primer pair were tested by 2% agarose gel electrophoresis to verify the products’ size and absence of non-specific bands. The qPCR efficiency of each gene was determined by using slope analysis with a linear regression model. Undiluted cDNA samples were used to calculate the PCR efficiency and correlation coefficient (R²) for each primer pair based on the standard curve method. The standard curve was generated with five-fold serial dilutions of cDNA. The corresponding qPCR efficiencies (E) were calculated according to the equation E (%) = (10^(−1/slope) − 1) × 100⁴³. Amplicons from the newly designed primers were Sanger sequenced and aligned with transcriptome data to confirm identity of the product.

Description of primers used in this study for RT-qPCR analysis.