Notch-1 and -2 was screened for mutation by using single stranded conformation analysis. PCR was performed to amplify the exons 26, 27 and 34 of the Notch-1 and -2 genes with 9 different primer pairs. Radiolabeling was done for primary PCR fragment with32P-nucleotide in secondary PCR reaction.  Labeled product were diluted to 1:20 with denaturing loading solution and denatured at 95°C for 3 min and immediately loaded on 15% polyacrylamide gel containing 10% glycerol. The fragments were separated at 6W constant power at room temperature (RT) for 16-18h.

Quantitative real-time PCR were performed using the TaqMan Copy Number Assay. Pre-designed probe for Notch1  and Notch2 were used for CNV assays for regions on 9q34.3d and 1p12a, respectively. RNase P was used as reference gene, which is always present in two copies. qPCR was performed in triplicates; each 10µl reaction volume, comprised of 10ng of DNA, 0.5µl of TaqMan primer/ probe, 0.5µl of RNase P primer/ probe and, 5µl of TaqMan universal Master Mix. Amplification was carried on Applied Bio systems 7900HT SDS.

Univariate analysis was carried out with log rank test, and Kaplan-Meier curves were generated for Overall survival analysis. Overall survival was defined as the time elapsed from diagnosis to the date of death. Statistical analysis was carried out with STATA v10.1.  P-value <0.05 was considered as statistically significant.