Multicolour real time pcr detection system

Total RNA was extracted from different tissues (midgut, fat body, haemocytes, integument, Malpighian tubules, head and silk gland) and developmental stages (egg to adult) using Trizol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The concentration and integrity of RNA were observed at an absorbance ratio of A_260/280 and A_260/230 using a NanoDrop 2000 spectrophotometer (Thermo Scientific^TM, Waltham, MA, USA), and by 1.0% agarose gel electrophoresis, respectively. Total RNA samples were used to prepare first strand cDNA using PrimeScript^TM RT Master Mix (Takara, Dalian, China) following the manufacturer’s instructions. The qRT-PCR was performed with a TB Green kit (Takara) and analyzed with the Multicolour Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), as previously reported [36 (link)]. The thermal cycling profile was set as follows: initial denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 20 s. The transcriptional level of Bmserpin2 was normalized to B. mori GAPDH. The relative expression levels were calculated using the 2^−∆∆Ct method according to a previous protocol [37 (link)]. The experiment was conducted in triplicate.

RT-qPCR was performed to examine the expression levels of ScFerHCH in various tissues and under different post-treatments. The primers used in this study were designed by Primer Premier 5.0 (Premier Biosoft, www.premierbiosoft.com) (Table 1). The 25 μL reaction mixture for the RT-qPCR contained 12.5 μL SYBR II, 9.5 μL ddH₂O, 1.0 μL forward primer, 1.0 μL reverse primer and 1.0 μL cDNA template. The thermal cycling profile consisted of an initial denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 20 s. The reactions were conducted in 96-well plates with a Multicolour Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). Relative expression levels were calculated using the 2^−∆∆Ct method [58 ]. There were three biological sample replicates, and each biological sample replicate included three technique replicates. The reference gene was S. c. ricini β-actin. The statistical analysis was conducted using ANOVA and an LSD a posteriori test using SPSS (p < 0.05).