Cfx maestro software version 2

The expression levels of inflammatory markers in the ears of the mice were measured using qRT-PCR. Total RNA in the lymph nodes and skin tissue was isolated using Trizol^® (Invitrogen, Waltham, MA, USA). The RNA was reverse transcribed into cDNA using MMLV reverse transcriptase (Promega, Madison, WI, USA). Thunderbird Next SYBR qPCR Mix was used for qRT-PCR on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). The PCR program consisted of a cycle at 95 °C for 4 min, 40 cycles at 95 °C for 30 s, 57 °C for 30 s, and the last at 95 °C for 30 s. With the help of CFX Maestro Software version 2.3 (Bio-Rad Laboratories, Hercules, CA, USA), the obtained data were analyzed using the 2^−∆∆Ct method. The primer sequences are listed in Table 1. The results were normalized to GAPDH gene expression levels [35 (link)].

CTK’s Aridia^® COVID-19 Real-Time PCR Test packaging contains a COVID-19 PCR mix, COVID-19 positive and negative controls and PCR-grade water is also provided to reconstitute the lyophilized components. The COVID-19 PCR mix contains all real-time PCR components including DNA polymerase, reverse transcriptase, primers, probes, and dNTPs. For the RT-PCR, 15 µL of PCR mix and 5 µL of RNA sample template, positive control and negative control were mixed to target the ORF1ab, N, E, and a housekeeping gene RNase P. PCR was performed according to the manufacturer’s instruction. A Bio-Rad CFX Opus96 Real-Time PCR System was used for the amplification and the thermocycler was programmed as followed: reverse transcription (10min, 50 °C, one cycle), (2 min, 95 °C, one cycle), denaturation (5 s, 95 °C, 45 cycles) and finally, annealing/extension (20 s, 60 °C, 45 cycles). At the extension step, fluorescence data are collected through HEX, JOE or VIC (ORF1ab), FAM (N gene), Cy5 (E gene), and ROX (RNase P) channels by Bio-Rad CFX Maestro Software Version 2.2. When N, ORF1ab and E genes showed cycle threshold (Ct) < 40, samples were considered positive and when ≥40, samples were considered negative for COVID-19.

RNA was isolated from hippocampi dissected and homogenized in TRIzol™ (ThermoFisher Scientific, Milan, Italy). RNA yield was determined with the Qubit Fluorometer (ThermoFisher Scientific). After DNase digestion, RNA was retrotranscribed using the iScript Clear cDNA Synthesis kit (Bio-Rad, Coralville, IA, USA). qPCR analysis was performed in PrimePCR^TM “Neurogenesis Tier 1 M96” collection panel (Bio-Rad), a predesigned 96-well PCR plate containing primer sets for 88 gene targets involved in neurogenesis pathway for use with SYBR^® Green (Table S2) on a CFX Connect Real Time System (BioRad). Ct values > 35 were considered to be no expression. Fold change calculation by ΔΔCt method [60 (link)] was performed with the CFX Maestro™ Software version 2.2 (Bio-Rad, Hercules, CA, USA) using three reference genes for normalization: TATA-binding protein (Tbp), glyceraldehyde-3-phosphate dehydrogenase (Gadph), and Hypoxanthine-guanine phosphoribosyltransferase (Hprt1).

Probit analysis was used to calculate LD₅₀ of spinosad for SLP, OFP and CFP populations [58 ]. The populations that had no overlap between the 95% fiducial limits of LD₅₀ were considered to have different resistance levels to spinosad as described previously [59 (link)]. RT-qPCR results were analyzed using the 2^-ΔΔCt method utilizing Bio-Rad CFX Maestro software version 2.2, while the statistical analyses of the data were done using two sample T-tests or one-way ANOVA followed by Tukey’s HSD post hoc analysis where applicable, using R version 4.3.1 [60 ]. Finally, Kaplan-Meier survival curves and Log-rank tests were performed using survival package in R to identify differences in survival in insects exposed to different treatments. A P-value of ≤ 0.05 was used as significance level in all analyses.