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.