Brightgreen qpcr mastermix

Total RNA was isolated from the HMC3 cells with a Trizol LS Reagent Kit (Life Technologies, Milan, Italy) and then quantified with a NanoDrop Lite spectrophotometer (Thermo Fisher, Milan, Italy). A total of 1 μg of total RNA was reverse-transcribed in a final volume of 20 μL by using the Superscript IV RT Master Mix (Invitrogen, Carlsbad, CA, USA). A total of 1 μL of cDNA was added to the BrightGreen qPCR Master Mix (ABM, Richmond, BC, Canada) together with specific primers at the concentration of 10 μM in a total volume of 20 μL/well to evaluate IL-1β, IL-6, Il-10, NLRP3, Caspase-1, and TNF-α mRNA expression. A qPCR reaction was monitored by using the QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher, Milan, Italy); GAPDH was used as housekeeping gene and the amplified PCR products were quantified by measuring the cycle thresholds (CT) of the target genes and GAPDH. After normalization, the mean value of the control group was chosen as the calibrator and the results were expressed according to the 2^−ΔΔCt method, as a fold change relative to the calibrator [29 (link),30 (link),31 (link),32 (link)]. Primers used for targets and reference genes are listed in Table 1.

At the end of the treatment period, differentiated SH-SY5Y and HMC3 cells were scraped employed TRIzol RNA Isolation Reagents (Thermo Fisher Scientific, Waltham, MA, USA), total RNA was extracted from and quantified by UV-Vis microvolume spectrophotometer (NanoDrop Lite, Thermo Fisher, Waltham, MA, USA). A Superscript IV Master Mix (Invitrogen, Carlsbad, CA, USA) was used to reverse transcribe the total RNA (1 µg) in cDNA. The obtained cDNA (1 μL) was added to the BrightGreen qPCR Master Mix (ABM, Richmond, Canada) to assess the mRNA level of IL-1 β and IL-6. qPCR reaction was monitored by using the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, CA, USA), the GAPDH gene was used as the housekeeping gene, and the amplified PCR products were calculated by measuring the calculated cycle thresholds (CT) of target genes and GAPDH mRNA. After normalization, the mean value of the normal control target levels was chosen as the calibrator and the results were expressed according to the 2^−ΔΔCt method, as a fold change relative to normal controls [42 (link),43 (link),44 (link),45 (link)].

To confirm the antiviral efficacy of M, H, and EA, a quantitative polymerase chain reaction (qPCR) was performed. The tests were conducted as described above. After incubation, the collected cells were subjected to total RNA extraction with TRIzol (Thermo Fisher, Waltham, MA, USA). The obtained RNA was quantified using the nanodrop (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA) and 1 microgram was retrotranscribed in cDNA, according to the instructions of the SensiFAST ™ cDNA Synthesis Kit (Meridian Bioscience, Washington, DC, USA). The qPCR was performed in a total volume of 20 µL containing 0.3 µM of each primer, 1X BrightGreen qPCR MasterMix (abm, San Francisco, CA, USA), and 100 ng of cDNA. Amplification was conducted in Thermal Cycler UNO96 (VWR International, Radnor, PA, USA) using the following amplification program: denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 15 s (40 cycles). The expressions of UL27 and UL54 (for HSV-1) and protein S and N (for HCoV-229E) were evaluated using the primer sequences reported in Table 2. The target threshold cycle (Ct) values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as a housekeeping-gene control. Gene expression was examined by calculating 2^−ΔΔCt.