4.5. Analysis of Cell Cycle by RT2 Profiler PCR Array for Cell Cycle

The Human Cell Cycle RT2 Profiler PCR Array (PAHS-0820, SABiosciences, Qiagen, Italy) was used to evaluate the expression of 84 specific genes related to the cell cycle. After 24 h of culture with rhIL-33 (10 ng/mL) and without any challenge, total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Milan, Italy) according to the manufacturer’s instructions. cDNA was synthesized from 500 ng of the total RNA using the RT2 First Strand Kit (Qiagen, Milan, Italy). PCR arrays were performed in 96-well plates on a StepOne Plus instrument (Life Technology, Monza, Italy). Briefly, the reaction mix was prepared from 2 × SABiosciences RT2 qPCR Master Mix and 102 µL of sample cDNA. Then, 10 µL of this mixture was added to each well of the PCR array. The thresholds and baselines were set according to the manufacturer’s instructions, and data were analyzed using software supplied by Qiagen (http://www.sabiosciences.com/pcr/arrayanalysis.php, accessed on 23 November 2020). The fold change in gene expression was calculated using the ΔΔCt method. More than 1.5-fold change and p < 0.05 in gene expression compared to unstimulated control were considered the up- or dowregulation of a specific gene expression. The gene pathway modified by IL-33 was reconstructed by IPA Software (SABiosciences, Qiagen, Milan, Italy). The genes differentially expressed in the two cell lines were tested by qRT-PCR after total RNA extraction from cells under various culture conditions using the RNeasy Kit (Qiagen, Milan, Italy), following the manufacturer’s instructions.