2.2. Sample Collection, RNA Extraction, and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

Blood collection, RNA extraction, and RT-qPCR were performed as previously described [26]. Approximately 3 mL of peripheral blood was collected in PAXgene blood RNA tubes (Qiagen, Hilden, Germany) and incubated at room temperature for 2 h. RNA was extracted using an RNA extraction kit (PAXgene Blood RNA Kit; Qiagen). RNA samples were evaluated using a NanoDrop spectrophotometer (ND-1000; Thermo Fisher Scientific, Waltham, MA, USA) and treated with DNase (TURBO DNA-free Kit; Ambion, Austin, TX, USA) to remove contaminating genomic DNA. Reverse transcription was performed using 1 µg of total RNA with random primers and a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The reverse transcription cycle was performed at 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 s in a thermal cycler. PCR analysis was performed using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the ABI7300 real-time PCR system (Applied Biosystems), as previously described [24]. The primers used to amplify each gene are listed in Table 1 [24]. The amplification conditions were as follows: initial sample incubation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min, with the collection of fluorescence signals at the end of each cycle. The melting curve for detecting the SYBR Green-based objective amplicon was confirmed at 65 to 95 °C in 0.5 °C increments. To quantify the concentration of each mRNA, standard curves were generated via the serial dilution of a plasmid containing the corresponding cDNA, as previously described [22]. For standard plasmid preparation, PCR fragments were subcloned into the pGEM-T Easy Vector System (Promega, Madison, WI, USA), according to the manufacturer’s instructions, and the sequences of the cloned plasmid containing cDNA were analyzed using the ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). In addition, the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for the normalization of internal expression in the samples. The expression of beta-actin and ribosomal protein L27, in addition to that of GAPDH, was also examined to evaluate their use as reference genes for RT-qPCR. The results showed no significant differences in their expression. Quantitative PCR was performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [35].

Primer sequences for RT-qPCR.