2.4. RNA isolation, reverse transcription, and quantitative PCR
analysis
Villous explants were collected from culture after exposition to
pro-inflammatory cytokines and stimulants for 24h and 72h, placed to
TriReagent (Molecular Research Center, Cincinnati, OH, USA) and stored
in -80°C until isolation of RNA. Subsequently the reverse transcription
was performed using RevertAid RT Reverse Transcription Kit (Thermo
Fisher Scientific) following the manufacturer´s instructions.
Analogically the cDNA for the qRT-PCR analysis was obtained from BeWo
and HTR-8/SVneo cells cultured under inflammatory environment. The
obtained cDNA samples were further evaluated by PCR using TaqMan®
Universal Master Mix II (Thermo Fisher Scientific) and predesigned
TaqMan® Real Time Expression PCR assays for human SLC22A4(Hs00268200_m1), SLC22A5 (Hs00929869_m1) (Applied Biosystems,
Thermo Fisher Scientific USA) and then analyzed using the QuantStudio™ 6
system (Thermo Fisher Scientific). Each sample was amplified in
duplicate using the following PCR cycling profile: 95 °C for 10 min,
followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. Samples
were measured in duplicate and normalized to the housekeeping geneYWHAZ (Hs01122445_g1). A non-template-control (NTC) was included
as a negative control. Value of ∆Ct
was calculated for all samples as a difference between Ct value of
target OCTN gene and Ct value of housekeeping gene (∆Ct =
CtOCTN – CtYWHAZ). The level of
expression of each gene was calculated by the 2− ΔΔCTmethod. Values of ∆∆Ct in samples of cells and cultured explants exposed
to inflammatory agents, were calculated as ∆∆Ct = ∆Ct -
∆Ctref, where ∆Ctref is determined from
an untreated control using YWHAZ as the reference gene.