Ssoadvanced universal sybr green supermix

Eleven CYP genes previously described by Giraudo et al.⁴² (link) and Nascimento et al.¹³ to be involved in insecticide detoxification were investigated in Sf_Bra and Sf_Des strains by RT‐qPCR. The ribosomal genes rsp3A, L17, and L10 were used as reference genes (primers and accession numbers of all genes are listed in Table S2). Reactions were performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio‐Rad, USA) according to the manufacturer's protocol. Briefly: reaction mixtures (10 μL) contained 2.5 μL cDNA (5 ng), 5 μL SsoAdvanced™ Universal SYBR® Green Supermix (Bio‐Rad), 400 nm of reverse/forward primers (Table S2), and nuclease‐free water. Reactions were run in triplicate using CFX384™ Real‐Time system (Bio‐Rad) and nontemplate mixtures as negative controls. The PCR conditions were: 3 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. A final melting‐curve step was included post‐PCR (ramping from 65 °C to 95 °C by 0.5 °C every 5 s) to check for nonspecific amplification. Amplification efficiencies were determined by a five‐fold dilution series revealing for all primers an efficiency ≥93%.

GFP positive HEK 293T cells, transfected with either pEGFP or pEGFP-E1, were sorted by flow cytometry (BD FACSAria™ II, USA), and DNA, RNA, and protein from the sorted cells were extracted using NucleoSpin® TriPrep (Macherey-Nagel, Germany). Extracted RNA (3 μg) was reverse transcribed using Super Script IV (Invitrogen, USA) according to manufacturer protocol. The cDNA from pEGFP or pEGFP-E1 transfected cells was amplified by real-time PCR using PrimePCR™ 96 well as follows: 10μL of 2x SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, USA), 1μL of cDNA template, and 9μL of Nuclease-free H₂O for a final volume of 20μL. The PCR reaction was as follows: initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds. GAPDH was used as a reference gene. Gene expression analysis was calculated using the 2^-ΔΔCT method.

qPCR analysis was performed using the 2x SsoAdvanced Universal SYBR Green Supermix as well as the PrimePCR SYBR Green Assays (both Bio-Rad, Munich, Germany) containing unlabeled oligonucleotide primer pairs and were used according to the supplier’s instructions. Amplification was carried out in a reaction volume of 20 μl using cDNA generated from 10 ng of RNA on the Bio-Rad C1000 Touch Thermal Cycler (CFX Manager Software Version 3.0; Munich, Germany). The thermal cycling protocol included a single polymerase activation step at 95°C for 2 minutes followed by 40 amplification cycles as well as melt-curve analysis. Each amplification cycle comprised a denaturation step at 95°C for 10 seconds and a primer annealing/elongation step at 60°C for 30 seconds. Melt-curve analysis was performed including a 30-second hold at 65°C followed by a gradual increase to 95°C with a temperature increment of 0.5°C every 5 sec. Primer efficiency was determined by performing standard curve analysis. Furthermore, intra-assay variation (repeatability) and inter-assay variation (reproducibility) were assayed. According to the minimum information for publication of qPCR experiments (MIQE) guidelines [6 (link)], all information including RNA, cDNA and qPCR analyses (e.g. conditions, reagents, quality rules) were summarized within the MIQE-checklist (S1–S3 Tables).

Total RNA was extracted using TRIzol reagent (Invitrogen), followed by phenol-chlorophorm extraction and isopropanol precipitation. After ethanol washes, the RNA pellet was air-dried, dissolved in nuclease-free water (Fermentas) and treated with RNase-free DNase (Roche) to remove possible genomic DNA contamination. Reverse transcription of 2 μg of total RNA to single-stranded cDNA was performed using random primers and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Next, the generated cDNA was diluted (1:20) and amplified using gene specific primers (Supplementary Table 1) and 2x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in HardShell^® Low-Profile Thin-Wall 96-Well Skirted PCR Plates (Bio-Rad) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad) under cycling conditions according to the manufacturer’s protocol. The relative expression levels were quantified using the comparative Cq method (ΔΔCq) with CFX Maestro software (Bio-Rad). Transcripts were normalized against 18s housekeeping gene that was experimentally determined to have the most stable expression in our reaction conditions.