qRT-PCR expression study
The expression level of transcription of some selected genes involved in the bacteria/osteoblast interaction. The primers, reported in table 1, were designed by the Flexi® Vector Primer Design Tool. Real Time PCR experiments on total RNA extracted from infected cell cultures, was carried out after 3h and 24h p.i.
RNA extraction was performed using the RNeasy mini-kit (Cat No.74104, Qiagen, Milan, Italy) following the manufacturer’s instructions, with some modifications. Briefly, bacteria internalized in MG-63 at 3h and 24h p.i. were collected in 500ul in RNAprotect Bacteria buffer (Cat No.76506, Qiagen) this first reaction series was carried out at a controlled temperature of about 4°C, vortexed and incubated for 5 min at room temperature. After centrifugation the pellet was resuspended in 100 ul of TE buffer containing: 20ul QIAGEN Proteinase K (Cat No.9131, Qiagen), lysozyme (cat No. 10837059001 Sigma-Aldrich-Merck KGaA, Darmstadt, Germany)150 mg/mL and lysostaphin (cat. No. L7386-15MG, Sigma-Aldrich-Merck KGaA) 20 mg/mL. After these changes, the indications provided by the manufacturer were followed.
The RNA quality was tested by Qubit® 3.0 Fluorometer (Cat No. Q33216, life Technologies, Thermo Fisher Scientific Monza, Italy) using Qubit RNA HS Assay Kit (250 pg/µl and 100 ng/µl). The RNA was normalized at 100 ng, to obtain the cDNA, using the QuantiTect reverse transcription kit (Cat No. 205311, Qiagen) and the amplifications were performed using QuantiTect Syber Green PCR Kit (Cat. No.204145, Qiagen) using a cDNA final concentration of 25ng/ul and 2uM primers per PCR reaction. Each sample amplification consisted of a total reaction volume of 10μL (5μL PCR Master Mix + 1μL specific primers + 4μL of cDNA (25ng/ul). Reactions were run in triplicate using the following conditions: PCR initial activation step 15 min 95°C; denaturation 15s 94°C; annealing 30s 60°C; extension 30s 72°C, the acquisition of fluorescence was done for 50 Cycle. The negative control consisted of a reaction in the absence of cDNA (5 μL PCR Master Mix + 1 μL specific primers + 4 μL of Tris-EDTA buffer) indicated as NTC (no template control).
qPCRs were performed in a Light Cycler® 480 Real Time PCR System (Roche, Monza, Italy). PCR efficiencies, melting curve analysis and expression rate were calculated using the Light Cycler® 480 Software (Roche, Monza, Italy).gyr B primers were used as internal control.
The relative RNA expression level for each sample was calculated using the 2−ΔΔCT method (threshold cycle (CT) value of the gene of interest vs CT value of the housekeeping gene) (Fresta et al., 2020). For accurate gene expression measurements with qRT-PCR, the results were normalized to the gir B housekeeping gene.