**3. Results**

### *3.1. Selection of Reference Genes*

We had chosen to use the Reference Gene qPCR Panel BioRad with primers being designed and coated on the plate in lyophilized form for selected reference genes adequate for the analyzed material obtained from volunteers [26]. The housekeeping genes were: ACTB, RPL13A, B2M, RPLP0, G6PD, RPS18, GAPDH, TBP, GUSB, TFRC, HMBS, YWHAZ, HPRT1, PGK1, and IPO8. Additionally, the panel contained several internal controls ensuring the most reliable results. After the RT-PCR reaction, the analysis of the melting curves for the obtained amplicons was carried out in order to determine the quality of the reaction. In the next stage, the Ct values that were detected for individual genes were used for further analyses, which were carried out using the BioRad program (CFX Manager ™ Software), and the GeNorm program. As a consequence of the analysis, we decided to use the following reference genes: GADPH, HPRT1, and TBP. These genes had the lowest M value corresponding to the most stable gene expression in the sample tested. The analysis of Ct values using the geNorm program allowed for further refinement of this gene list and, in turn, only HRTP1 and GAPDH were used as the reference panel, as shown in Figure 1.

**Figure 1.** Chart showing the most (lowest M value) and least stable (highest M value) reference genes indicated by the program GeNorm.

Melt peak analysis demonstrated a single homogenous peak for all primer sets, including selected reference genes (Figure 2).

### *3.2. IL-18, IL-18BP, IL-18R, IFN-*γ*, and IL-37 Gene Expression in the Studied Groups*

A significantly higher relative level of IL-18 mRNA expression was observed in LTB individuals as compared to healthy controls without *M.tb* infection (*p* = 0.023). A similar increase in the relative IL-18 expression level was observed among ATB patients; however, the di fference in values for the group ATB and group Control group was not significant (*p* = 0.082) (Figure 3A).

The relative level of IL-18BP mRNA expression was significantly higher in ATB patients (*p* < 0.001) and healthy LTB individuals (*p* = 0.006) than in Control group volunteers (Figure 3B).

There were major significant di fferences between the three groups in the IL-18R mRNA expression. In the LTB groups, the relative level of IL-18R mRNA was much lower than in the ATB patients (*p* < 0.001) and individuals from the Control group (*p* < 0.001) (Figure 3C).

The level of relative expression of IFN-γ mRNA was significantly higher in active TB patients (*p* = 0.002) and LTB individuals (*p* = 0.029) than in the Control group (Figure 3D).

*Pathogens* **2020**, *9*, 451

No statistically significant differences were observed in the levels of relative expression of IL-37 mRNA among the studied groups (Figure 3E).

**Figure 2.** Melt peak analysis of reference genes HPRT1 (light grey) and GAPDH (dark grey).

**Figure 3.** Relative expression of IL-18, IL-18BP, IL-18R, IFN-γ, and IL-37 mRNA in studied groups. Dot plot with mean (horizontal line), and standard deviation (whiskers), IL-18 (**A**), IL-18BP (**B**), IL-18R (**C**), IFN-γ (**D**), IL-37 (**E**) in the groups of healthy volunteers (Control), patients with active tuberculosis (ATB), and latently infected individuals (LTB).
