Page 117 - วารสารการแพทย์แผนไทย ปีที่ 21 ฉบับที่ 2

J Thai Trad Alt Med Vol. 21 No. 2 May-Aug 2023 333

solution and reagents were prepared following protocol, RNA was re-suspended in 30 µL of
the procedure described in the manufacturer’s nuclease-free water and the products ran on

protocol. The plant extracts were diluted with agarose gels to check the quality of the RNA.
water at the highest concentration, which was cDNA was synthesized using QuantiTect Rev.
not toxic to Calu-3 cells and then diluted to Transcription Kit (Qiagen, Valencia, CA, USA)

lower 2 concentrations. A Total of 30 µL of TM- following the manufacturer’s protocol. Briefly,
PRSS2 enzyme at a concentration of 5 ng/µL in 2-µg template RNA was added to the reverse-
1x TMPRSS2 Assay Buffer was added to 10 µL transcription master mix and then the samples

of diluted extracts or 10 µM camostat standard tubes were incubated at 42 ˚C for 15 min. The
inhibitor (the background and enzyme controls cDNA samples were tested in triplicate with
were also tested). The mixed solutions were quantitative PCR using a QuantiTect SYBR

placed at room temperature for 30 min. A total Green PCR Reagents kit (Qiagen, Valencia,
of 10 µL of TMPRSS2 fluorogenic substrate CA, USA). A teetotal of 2 µL of each sample

mix (50 µM) was then added to all wells and was mixed with SYBR Green PCR Master Mix
placed at room temperature for 10 min whilst and 10x QuantiTect Primers (Qiagen, Valencia,
being protected from light. The plate was im- CA, USA), then performed the real-time PCR

mediately placed in a spectrofluorometer and (RT-qPCR) followed the manufacturer’s pro-
the fluorescent signal measured (Ex/Em = tocol in Thermal cycler (PCR) (Analytik Jena

383/455 nm) in kinetic mode for 30 min. The GmbH, Valencia, Jena, Germany). mRNA ra-
relative inhibition activity (%) of the sample tios relative to the glyceraldehyde 3-phosphate
was calculated using a relative fluorescence dehydrogenase (GAPDH) housekeeping gene

unit (RFU) compared to enzyme control. were calculated for the standardization of gene
2.6 Gene expression using real‑time expression levels. A melting curve analysis
reverse‑transcription polymerase chain reac‑ was also performed to verify the specificity and

tion (RT‑PCR) identity of PCR products. For selected genes,
5
Calu-3 cells were seeded at 5 5 10 cells/ the data were analyzed using the equation
[20]
well in 6-well plates for 24 hours, then treated described by Livak and Schmittgen as fol-
with the compounds or extracts at concen- lows: the amount of target =2 − ∆∆Ct . The average
trations that were not toxic to the cells for 24 ∆Ct from the untreated cells is a calibrator for

hours. Total RNA of each sample was extract- each gene tested. This assay was conducted
ed using the RNeasy Mini Kit (Qiagen, Hilden, at Toxicology laboratory, Medicinal Plant
Germany). According to the manufacturer’s Research Institute, Department of Medical