Brightgreen 2 qpcr mastermix no dye

The virus pre-treatment assay was chosen for the molecular test and performed as previously reported at the same volumes (from 100 to 12.5 µL), and after only 1 h stimulation. After 48 hpi, RNA was extracted by using the TRIzol reagent (Thermo Fisher, Waltham, MA, USA). Total RNA was quantified through the absorbance at 260 nm (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA) and retrotranscribed to cDNA by 5× All-In-One RT Master Mix (Applied Biological Materials, Richmond, VA, Canada). Real-time PCR was performed using a CFX thermal cycler (Bio-Rad, Hercules, CA, USA) and amplified through BrightGreen 2× qPCR MasterMix-No Dye (Applied Biological Materials, Richmond, VA, Canada) and 0.1 µM of primer. The relative target threshold cycle (Ct) values of UL54 (immediate early gene), UL52 (early gene), and UL27 (late gene) were normalized using glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as the housekeeping gene. The mRNA levels of cells treated with ophthalmic solutions were calculated via the 2^−∆∆Ct method. The primer sequences and the thermal conditions are shown in Table 1.

Vero were plated into 12-well cell culture plates (2 × 10⁵ cells/mL cells for each well) in culture medium. The next day, the monolayer was treated with leaf extract and HSV-1 and SARS-CoV-2, as described above. After 24 h, the total RNA was isolated using TRIzol reagent and quantified by measuring the absorbance at 260/280 nm (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA). A total of 1 µg of total RNA was reverse transcribed to cDNA by 5× All-In-One RT MasterMix (Applied Biological Materials, Richmond, Canada). A quantitative polymerase chain reaction was run in triplicate using a CFX Thermal Cycler (Bio-Rad, Hercules, CA, USA). Then, 2 µL of cDNA were amplified in 20 µL reactions using BrightGreen 2× qPCR MasterMix-No Dye (Applied Biological Materials) and 0.1 µM of primer. Relative target Ct (the threshold cycle) values of UL54, UL52 and UL27 (for HSV-1), and S protein (for SARS-CoV-2) were normalized to GAPDH, as the housekeeping gene. The mRNA levels of cells treated with leaf extract were expressed using the 2^−ΔΔCt method [51 (link)]. Primers were purchased by Eurofins (Vimodrone, Milan, Italy) and their sequences are reported in Table 1.

The total RNA of the SK-MEL-2 cells was isolated using TRIzol^® Reagent (15596026, Invitrogen, Waltham, MA, USA), and cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems, Foster City, CA, USA). The tyrosinase inhibition rate in the SK-MEL-2 cells was determined using a tyrosinase Activity Assay Kit (MAK395, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s manual. The tyrosinase mRNA level was measured using BrightGreen 2× qPCR MasterMix-No Dye (Applied Biological Materials, Richmond, BC, Canada), according to the supplier’s manual. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control, and the relative gene expression levels were analyzed using the 2^−ΔΔCt method. Primers for GAPDH (forward 5′-GAGTCAACGGATTTGGTCGT-3′, reverse 5′-GATCTCGCTCCTGGAAGATG-3′) and TYRP1 (forward 5′- ATGGCAGAGATGATCGGGAG-3′, reverse 5′-GAGCTTCAACTCCAACCCTT-3′) were used for cDNA amplification [36 (link)]. The thermocycling conditions were: 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s.