4.3. Relative mRNA Expression

Frozen ChP samples were homogenised with using FastPrep24 instrument (MP Biomedicals, Illkirch-Graffenstaden, France) and dedicated to Lysing Matrix D tubes (MP Biomedicals, Illkirch-Graffenstaden, France), filled with lysing buffer (RA1) from the NucleoSpin RNAII Kit (MARCHEREY-NAGEL, Düren, Germany) for total RNA isolation. According to the manufacturer’s protocol, during RNA isolation, the genomic DNA digestion step was carried out. The concentration and purity of the obtained RNA were evaluated spectrophotometrically by using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was controlled by electrophoresis method with using 1.2 % agarose gel containing the GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA USA). The reverse transcription (RT) of total RNA was performed using DyNAmo cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer protocol and each RT reaction contained 1µg of total RNA. Obtained cDNA was frozen and kept at −20 °C until further analysis.

For the real-time PCR analysis, the specific primers pairs were selected based on the literature data or were originally designed using Primer-BLAST (National Center for Biotechnology Information, Bethesda, MD, USA). All primers were synthesised by Genomed (Warsaw, Poland) and their sequences and source data are listed in Table 1.

Sequences of oligonucleotide primers used for real-time PCR.

LEPRa—leptin receptor short isoform; LEPRb—leptin receptor long isoform; LEPR—leptin receptor all isoforms; SOCS3—suppressor of cytokine signalling 3 (originally designed/1 coding exon); TLR4—Toll-like receptor 4; IL1B—interleukin 1-beta; IL1R1—interleukin 1 receptor, type I; IL1R2—interleukin 1 receptor, type II; IL1RN—interleukin 1 receptor antagonist; NLRP3—NLR family pyrin domain containing 3; PYCARD—PYD (pyrin domain) and CARD (caspase activation and recruitment domain) domain-containing; CASP1—caspase 1; IL6—interleukin 6; IL6R—interleukin 6 receptor; IL6ST—glycoprotein 130; CCL2—C-C motif chemokine ligand 2 (originally designed/F:149/150 exon span); * reference genes: GAPDH—glyceraldehyde-3-phosphate dehydrogenase; ACTB—beta-actin; HDAC1—histone deacetylase1.

The real-time PCR analysis was performed using the Viia7 instrument (Applied Biosystems by Life Technologies, Waltham, MA, USA) and each real-time PCR reaction contained 3 µL cDNA (1:10), 0.2 µM of each oligonucleotide from a specific pair and 5 µL of DyNAmo SYBR Green qPCR kit with ROX (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA samples and reaction mix were transferred into 384-well plates, by the Bravo Automated Liquid Handling Platform (Agilent Technologies, Santa Clara, CA, USA). The following protocol was used: 95 °C for 10 min for the hot start modified Tbr DNA polymerase, and 40 cycles at 95 °C for 15 s (denaturation), 60 °C or 55 °C for 30 s (primer annealing), and 72 °C for 30 s (extension). After the cycles, a final melting curve analysis was performed under continuous fluorescence measurement to assess the specificity of the amplification. The primer annealing temperature was evaluated empirically by gradient PCR analysis (Labcycler SensoQuest, Göttingen, Germany) and for almost all primer pairs was set at 60 °C and only for TLR4 at 55 °C.

Three housekeeping genes were examined: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB) and histone deacetylase 1 (HDAC1) using free NormFinder software (MOWA, Aarhus, Denmark). For the presented experimental conditions, we identified HDAC1 as the best reference gene, and HDAC1 and ACTB as the best combination of two genes, which was used in further analyses. Expression analysis was performed with using the Real-Time PCR Miner (available online: http://ewindup.info/miner/), a software based on the Zhao and Fernald [78] algorithm, that allows considering the actual reaction efficiency for each pair of primers for every single reaction.