4.6. RNA Extraction, cDNA Synthesis (Reverse Transcription) and Quantitative Real-Time PCR (qPCR)

For RNA extraction and gene expression analysis, 2 × 10⁵ cells per well were seeded in 12-well plates one day prior to treatment. On the next day, cells were synchronized by medium change and treated with C. cardunculus or vehicle control for 48 h.

Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. Prior to the purification procedure, medium was discarded, and cells were washed with PBS and lysed directly in RLT Plus buffer (Qiagen, Hilden, Germany) supplemented with 2-Mercaptoethanol (AppliChem, Darmstadt, Germany). Genomic DNA was digested using gDNA eliminator columns provided with the kit (Qiagen, Hilden, Germany). RNA was eluted in 30 µL RNase-free water. The final RNA concentration was measured using a Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA was then stored at −80 °C until use. Then, 1 µg of total RNA was reverse-transcribed to cDNA with M-MLV reverse transcriptase (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), random hexamers (Thermo Fisher Scientific, Waltham, MA, USA) and dNTPs Mix (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR was performed using human QuantiTect Primer assays (Qiagen, Hilden, Germany) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad laboratories, Hercules, CA, USA) in 96-well plates. GAPDH was used as reference genes. The following primers were designed in-house:

MACC1: FW-TTCTTTTGATTCCTCCGGTGA, REV-ACTCTGATGGCA-TGTGCTG.

WEE1: FW- CCCCGCCACACAAGACCT, REV-GAGAGCAAACTCTTGGGCGTG.

The following QuantiTect Primer assays (Qiagen, Hilden, Germany) were used:

GAPDH: QT00079247; BMAL1: QT00011844; PER2: QT00011207; NR1D1: QT00000413; CRY1: QT00025067; MYC: QT00035406; HKDC1: QT00086359

The qPCR reaction and the subsequent melting curve were performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad laboratories, Hercules, CA, USA). A melting curve analysis was performed to detect potential unspecific amplification products. Cq values were determined using the regression method. The expression levels were normalized to those of GAPDH (ΔCT) and calibrated in relation to the respective control (ΔΔCT). Relative quantification was calculated using the 2^−ΔΔCt method. Biological and technical replicates were included in the analysis. The mean and the standard error of the mean were calculated.