Quantitative Reverse Transcription PCR (RT-qPCR)

Normal endometrial specimens (n = 12) from the healthy control group, and ectopic endometrium (n = 35) and eutopic endometrium (n = 35) from patients with endometriosis were used for RT-qPCR. Total RNA was isolated from tissues or cells by using TRIzol reagent and the concentration of RNA was determined. The RNA quality was assessed by Nanodrop. The RNA was used to synthesize cDNA using a reverse transcription kit. After the reverse transcription reaction, the advanced master mix was used for quantitative analysis. To analyze the accuracy of the PCR reaction, the melt curves were used. To evaluate the expression of genes, the 2-^△△Ct values were calculated using GAPDH as an internal control. GAPDH is known as the most common internal control in RT-qPCR and its expression can be detected in the tissues and cells used in this study. Therefore, we selected GAPDH as an internal control gene. The primers were listed below:

HMGB-1 forward: 5’- GCT CAG AGA GGT GGA AGA CCA-3’, and reverse: 5’- GGT GCA TTG GGA TCC TTG AA-3’; beclin-1 forward: 5’- CCA TGC AGG TGA GCT TCG T -3’, and reverse: 5’- GAA TCT GCG AGA GAC ACC ATC -3’; GAPDH forward: 5’- CCA TGC AGG TGA GCT TCG T -3’, and reverse: 5’- TGT CAT CAT ATT TGG CAG GTT T -3’.