Abi prism 7000 sequence detection instrument

In order to evaluate the antioxidant effects of the AH and AL flower extracts in vitro, Caco-2 cells were treated for 48 h with extract in aqueous solution (1–20 µg/mL). The dose with the maximum effect observed in the antioxidant reactions described above was chosen. Total RNA was then extracted from 5 × 10⁶ cells following a standard protocol [51 (link),60 (link),61 (link),62 (link),63 (link)]. RNA was quantified spectrophotometrically and 2 µg was back-transcribed using the SuperScript^® VILO ™ cDNA synthesis kit (ThermoFisher, Mumbai, India). An aliquot (3.5 µL) of cDNA was then amplified by real-time PCR.
TaqMan^® MGB (ThermoFisher) probes were used to evaluate the mRNA expression levels of genes involved in cellular oxidative processes. The probes were heme oxygenase-1 (HO-1) (Hs01110250_m1), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Hs00975961_g1), and NAD(P)H quinone reductase (NQO1) (Hs01045994_m1). The beta-actin gene (Hs01060665_g1) was used for housekeeping. All samples were amplified in triplicate in 96-well optical plates (ThermoFisher) by the ABI Prism 7000 Sequence Detection instrument (Applied Biosystems) following the instrument protocol. Quantification was performed using the comparative method C (T), also called method 2 (−ΔΔCT).

Whole blood samples were obtained prior to, during, and following treatment. During the treatment period, blood was collected 13–14 hours or at indicated time points after administration of AIC649 or vehicle. Total RNA was isolated using the QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Following DNase I treatment (Invitrogen, San Diego, CA) and reverse transcription with the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA) using oligo-d(T)16, cDNA was amplified by real-time PCR using 2X Core Reagent kit (Applied Biosystems) and the ABI PRISM 7000 Sequence Detection instrument (Applied Biosystems) as described previously [26 (link)]. Woodchuck-specific primer and probes for the detection of interleukin-2 (IL-2), IFN-γ, and TNF-α were used [26 (link)]. Expression of 18S rRNA (Applied Biosystems) was used to normalize target gene expression. Transcript levels of target genes were determined by the formula 2^ΔCT, where ΔC_T indicates the difference in the threshold cycle between 18S rRNA and target gene expression. Cytokine transcript levels were then calculated as fold change relative to pre-treatment (“day 0”).