Figure 2: Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis in 78 DFU cases and 8 controls. RNA was isolated from wound samples collected during debridement of DFU patients and controls, cDNA was synthesized and RT-PCR for TLR4 and GAPDH was performed. (A) Representative picture of controls {C} and DFU {P} in each group is shown. (B) Bar Graph represents the percent ratio which was calculated for the expression of TLR4 and GAPDH in Controls and DFU respectively. The percent ratio was determined by dividing the band intensity of TLR4 by GAPDH. Unpaired t test was used to check the difference between the mean values of TLR4 mRNA in DFU and control subjects. A two tailed p value < 0.05 was considered as statistically significant. Semi quantitative RT-PCR analysis shows the reduced expression of TLR4 transcripts in DFU patients (p value=0.02, t=2.28, mean change in percent ratio=0.99, Standard error of mean=0.04) (C) Representative graph showing Quantitative Real-time PCR analysis showing the reduced expression of TLR4 mRNA and VEGF in wounds of DFU patients. Analysis was done in randomly selected 43 DFU cases and 8 controls. Fold change in expression of genes was determined using the ΔΔCT method of relative quantification. Firstly normalization of the resulting threshold cycle (CT) values of the target gene was done with the CT values of the internal control GAPDH in the same samples (ΔCT=CT target – CT GAPDH). It was again normalized with the control (ΔΔCT=ΔCT – ΔCT control). The fold change in the expression was then calculated (2-ΔΔCT). The graph was plotted using log 2-ΔΔCT. The graph clearly showed that TLR4 was decreased significantly in the wounds of T2DM cases (p value=<0.0001, mean fold change=-1.13, Standard error of mean=0.17). Similar down regulation was showed by the VEGF mRNA (p value=< 0.0001, mean fold change=-1.39, Standard error of mean=0.10).