Applied biosystem 7500 real pcr system

The sequences of P. americana targeted genes aaNAT (GenBank: AB106562.1), Vg1 (GenBank: AB034804.1), Vg2 (GenBank: AB047401.1), VgR (GenBank: AB077047.2), JHAMT (GenBank: LC164750.1), FAMeT (GenBank: LC164751.1) and Actin (GenBank: AY116670.1) were retrieved from NCBI (data from our laboratory). The gene-specific primers were designed using Primer 3 Tool. RNAs and template cDNAs were prepared as mentioned above. qPCR was performed with the SYBR^® Green Realtime PCR master mix (Toyobo, Osaka, Japan), with the forward and reverse primers shown in Table 1. Cycling parameters were 95 °C for 1 min, 40 cycles programmed for 95 °C for 15 s and 60 °C for 2 min. The acquisition of fluorescence data was performed at the end of the elongation step with Applied Biosystem 7500 real PCR system (Applied Biosystems, Foster City, CA, USA). Initial amount of template was calculated from cDNA standard curve generated for each PCR run. Three independent biological replicates containing three pooled insects for each were run per condition. Relative transcript quantity was calculated using the C_T (ΔΔC_T) method [37 (link)]. Actin mRNA was used as the reference gene for all samples.

RNAs and template cDNAs were prepared as mentioned above. qRT-PCR was performed by using SYBER Green Realtime PCR master mix (Toyobo, Osaka, Japan) and Applied Biosystem 7500 real PCR system (Applied Biosystems, CA, USA). Actin of A. pernyi (accession no. GU176616) was amplified with gene-specific primers (Actin-F, Actin-R) (Table 2) and quantified for C_T in each sample as an internal control. Each treatment was replicated three times. Quantitative analysis followed by a comparative C_T (ΔΔC_T) method was used for analysis [44] (link). For each gene, the primers used in qRT-PCR were designed for the outer region of dsRNA, namely, primers for nat (NAT-F, NAT-R), cyc (CYC-F, CYC-R), clk (CLK-F, CLK-R) and per (PER-F, PER-R) (Table 2).