Stained sections were observed under a microscope. Immunostaining was scored by two independent experienced pathologists, who were blinded to the clinicopathologic parameters and clinical outcomes of the patients. An immunoreactivity score system was applied as described previously [12]. The extensional standard was: (1) the number of positively stained cells <5% scored 0; 6-25% scored 1; 26-50% scored 2; 51-75% scored 3; >75% scored 4; (2) intensity of stain: colorless scored 0; pallide-flavens scored 1; yellow scored 2; brown scored 3. Multiply (1) and (2). The staining score was stratified as – (0 score, absent), + selleck kinase inhibitor (1-4 score, weak), ++ (5-8 score, moderate) and

+++ (9-12 score, strong) according to the proportion and intensity of positively stained cancer cells. Specimens were rescored if difference of scores from two pathologists was >3. 2.3 Quantitative real-time PCR Total RNA purified from all 252 glioma tissues and 42 control brain tissues was prepared and reverse transcribed. Real-time monitoring of polymerase chain reactions (PCRs) was performed using the ABI 7900HT (Idaho Technology, Idaho Falls, ID, USA) and the

SYBR green I dye (Biogene), which binds preferentially to double-stranded DNA. Fluorescence signals, which Selleckchem LCZ696 are proportional to the concentration of the PCR product, are measured at the end of each cycle and immediately displayed on a computer screen, permitting realtime monitoring of the PCR. The Non-specific serine/threonine protein kinase reaction is characterized by the point during cycling when amplification of PCR products is first detected, rather

than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting quantity of the template, the earlier a significant increase in fluorescence is observed. The threshold cycle is defined as the fractional cycle number at which fluorescence passes a fixed threshold above the baseline. The primers 5′- TAT TAA GCA TGC TAT ACA ATC TG -3′ and 5′- CTT CCA CCC AGA TTT CAA TTC -3′ were used to amplify 332-bp transcripts of SMAD4 and the primers 5′- GGT GGC TTT TAG GAT GGC AAG -3′ and 5′- ACT GGA ACG GTG AAG GTG ACA G -3′ were used to amplify 161-bp transcripts of βselleck products -actin. All primers were synthesized by Sangon Co. (Shanghai, China). The PCR profile consisted of an initial melting step of 1 min at 94°C, followed by 38 cycles of 15 s at 94°C, 15 s at 56°C and 45 s at 72°C, and a final elongation step of 10 min at 72°C. Fluorescence data were converted into cycle threshold measurements using the SDS system software and exported to Microsoft Excel. SMAD4 mRNA levels were compared to β-actin. Thermal dissociation plots were examined for biphasic melting curves, indicative of whether primer-dimers or other nonspecific products could be contributing to the amplification signal. 2.4. Western blot analysis Glioma and normal brain tissues were homogenized in lysis buffer [PBS, 1% nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.