Sybr green 1 fluorescent dye

Corneal tissues of each group were taken out of the freezer 1, 3, 7, 14, and 28 days after alkali burning and total RNA was extracted using Trizol reagent (Invitrogen Inc., Carlsbad, CA, USA). A reverse transcription kit (Tiangen Biotechnology Co. Ltd, Beijing, China) was used to transcript total RNA into cDNA. The following specific experimental procedures were followed. SYBR Green I fluorescent dye (Applied Biosystems, Inc., Foster City, CA) was applied to conduct the qRT-PCR. The reaction system of each gene was 20 μl: 10 μl 2 × SYBR Green, 0.3 μl of upstream and downstream primers (20 μM), 1.0 μl cDNA, and 8.4 μl ddH₂O. The reaction conditions were: 95 °C for 5 min, 95 °C for 30 s, and 60 °C for 1 min (40 cycles in total). β-actin served as an internal reference. The primer sequences are shown in Table 1. The relative expression of S100A4, VEGF, and TNF-α in each group and at the different time points was performed using the following formula: n = (1+E)^-ΔΔCT [21 (link)]. E indicates the amplification efficiency of the target gene primer (E=10^{-1/Standard curve slope}) [22 (link)]. In this experiment, the primer amplification efficacy was calculated as ΔCT = (CT_{target gene}-CT_β-actin).

The six randomly selected DEGs were Janus kinase 3 (Jak3), cyclin-dependent kinase-1 (CDK1; also known as Cdc2a), Bcl2-like 2 (Bcl2l2), nuclear factor-κB (NF-κB), tumor necrosis factor receptor superfamily, member 1A (Tnfrsf1a), and SDH were detected by qPCR. PCR amplification was performed using an iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories, Richmond, CA, USA) with SYBR-Green I fluorescent dye (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primer sequences for Jak3, Cdc2a, Bcl2l2, NF-κB, Tnfrsf1a, SDH and 18S RNA are presented in Table I. The PCR program included 95°C for 10 min, 40 cycles at 95°C for 15 sec, 55°C for 1 min, and a final 20 min extension at 60–95°C. Finally, the expression of each gene was calculated using the 2^−ΔΔCq method (23 (link)) and 18S RNA was used as the internal control.

A SYBR Green I–based assay was used to measure in vitro P. falciparum drug susceptibility^{168 (link),169 (link)}. Ring-stage NF54 parasites were seeded at 1% hematocrit and 1% starting parasitemia in 384-well black clear-bottom plates containing test compounds (stocks of 10 mM pyrimethamine, 0.1 mM atovaquone, and 10 mM DHA in DMSO) plated in triplicate in 12-point serial dilutions for 72 h. The parasites were plated in complete RPMI 1640 media supplemented with 25 mM HEPES, 0.21% sodium bicarbonate, 50 mg/l hypoxanthine, and 0.5% Albumax II (Invitrogen) with or without 1 mM DAHP. Lysis buffer (0.16% w/v saponin, 1.6% Triton X-100, 5 mM EDTA, and 20 mM Tris-HCl, pH 7.4) with a 1:1000 dilution of SYBR Green I fluorescent dye (Invitrogen) was added for at least 12 h, and fluorescence readings were taken (excitation at 494 nm, emission at 530 nm) using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) plate reader. IC₅₀ values were calculated using a nonlinear regression curve fit in Graphpad Prism 9 in biological triplicate.