Analysis of polymorphisms in the OPN regulatory region:
The PCR amplification of the promoter regulatory region from the extracted DNA by using the following primers -3 (forward primer 5" -CAA GCT ACT GCA TAC TCG AAA TCA CA-3" ; reverse primer 5" - ACA ACC AAG CCC TCC CAG AAT TTA-3" ). PCR was performed using 50 ng DNA as a template under the following conditions: 95°C for 10 min, then 36 cycles of 94°C for 30 s, an annealing temperature for 60 s, and 72°C for 60 s, with a final extension at 72°C for 15 min. After affinity membrane purification using the QIAquick Gel Extraction kit (Qiagen, Carlsbad, CA, USA), the PCR products were subjected to cycle sequencing with the respective forward and reverse primer using an automated ABI 310 DNA sequencer.
Measurement of serum level of osteopontin:
OPN level were measured using Enzyme linked immnosorbent (ELISA) Kit supplied by IBL (Immuno-Biological laboratories Co. Ltd., Japan). 30 normal females were conducted with matched age with a median osteopontine serum level of 15 so, this level was assigned as a cut off value.
Pretreatment serum level and polymorphism of oteopontine was correlated with different clinicopathological criteria of the patients, response to the treatment, PFS and OS.
