Biofilm-related gene expression analysis by real-time QPCR

Bacteria collected from biofilm bacteria and planktonic bacteria with/without treatment were centrifuged for 10 minutes at 5000 g. RNeasy mini kit (Qiagen) was used to extract RNA from biofilm bacteria and planktonic bacteria following the manufacturer’s instructions. Total RNA was converted to cDNA using Superscript Vilo master mix (Invitrogen, USA). QPCR analysis was performed using 25 ng of cDNA and 1.25 μM of the appropriate primers using qPCRBio SYGreen master mix (PCR Biosystems, UK) and iQ5 real-time PCR detection system (Bio-Rad Laboratories, Hercules, California, USA). The genes involved in the formation/regulation of biofilms (rsbU, sarA, icaA, icaB, icaC and icaD), adhesin genes (embp and atlE) were analyzed and a house keeping gene tpi was used as control. The sequences of the primers used are listed in S1 Table. The experiments were carried out in triplicates with three independent repeats.