Cdna synthesis system

After exposure
to NTX and 6β-naltrexol, HUVECs and BeWo cells from the microfluidic
device were quantified using the RT-PCR method. After treatment, control
and experimental samples were trypsinized, pelleted, and frozen at
−80 °C, then integrated into single-control and single-experimental
sets before homogenization in TRIzol reagent (Invitrogen, ThermoFisher)
(trypsinized-HUVECs and -BeWo cells were perfused through channels
and collected from outlets separately). Following homogenization,
RNA isolation and reverse transcription were performed using the Absolutely
RNA Miniprep kit (Stratagene) and cDNA synthesis system (Applied Biosystems),
respectively. A Qiagen RT² SYBR Green master mix with validated
qPCR human primers (for HUVECs and BeWo) were used to determine relative
magnitudes of gene-expression levels using RT-PCR. Human 18S rRNA,
the housekeeping genes, were used to normalize each sample, and melting
curves and dissociation curves were constructed to verify the gathering
of nonspecific amplicons-free peaks, as described in manufacturer’s
recommended guidelines. The ΔC_t method
developed to utilize threshold cycle (C_t) values from housekeeping gene and respective gene was used to calculate
and report the results as a fold change in gene-expression.^{44 (link),45 (link)}

Total cellular RNA extraction was performed using an ultrapure RNA kit (Cwbiotech, Beijing, China) according to the manufacturer's protocol. Synthesis of complementary DNA was performed with a cDNA synthesis system (Applied Biosystems, Foster City, CA, USA). cDNA amplification was performed using a QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). β-actin was used as an internal control. The comparative Ct method (2^−ΔΔCt) was used to analyze data [28 (link)]. The sequences of primers for qPCR are as follows: β-actin forward：5′-GTATCCTGACCCTGAAGTACC-3’;

reverse: 5′-GAAGGTCTCAAACATGATCT- 3’;

HO-1 forward: 5′-TCTCTTGGCTGGCTTCCTTAC-3’;

reverse: 5′-GGCTTTTGGAGGTTTGAGACA-3’;

NQO-1 forward: 5′-TGCAGCGGCTTTGAAGAAGAAAG-3’;

reverse: 5′-TCGGCAGGATACTGAAAGTTCGC-3’.