Controls and patients were matched in age and gender. History reviewing, physical examination, and clinical examinations Imatinib Mesylate molecular weight were done in these controls and cerebrovascular diseases; other neurological diseases; kidney/liver diseases, hematological diseases, tumors, peripheral vascular diseases, and autoimmune diseases were excluded from these controls. There was no kinship among all these subjects who were Han Chinese in Liaoning Province, a region of North China. This study was approved by the Ethic Committee of China Medical University and 202 Hospital, and informed consent was obtained from all subjects before study. Questionnaire, physical examination, and laboratory examinations were performed to acquire clinical information of these subjects including age, gender, height, body weight, blood pressure, blood lipid, fasting blood glucose, past history, and history of smoking and drinking (Table 1).

Table 1Clinical information of IS patients and controls at baseline.2.2. GenotypingVenous blood (3mL) was collected from each subject and anticoagulated with EDTA. Genomic DNA was extracted with DNA extraction kit (Wizard Genomic DNA purification kit; Promega, USA). UV spectrophotometer was used to determine the concentration and purity of extracted DNA. The genomic DNA was stored at ?20��C.Four genetic loci of NOS1 (rs1483757, rs7308402, rs2293050, and rs2139733) were selected according to the gene sequence of NOS1 in dbSNP database of NCBI (http://www.ncbi.nlm.nih.gov/SNP/). SNaPshot multiple Minisequencing technology was used for genotyping [23].

First, genomic DNA underwent amplification by multiplex PCR with the following mixture (20��L): 1xGC buffer I, 3.0mM Mg2+, 0.3mM dNTP, 1 U of HotStarTaq polymerase (Qiagen Inc.), 1��L of DNA, and 1��L of multiplex PCR primers. The primers were as follows: rs1483757F: 5��-TGCCTCCGACAACTGAGCTGAT-3�� rs1483757R:5��-GCCTGCGTGACAGAGTCAAATTC-3�� rs7308402F: 5��-GCAGGCTTATCCCATGGCTCTT-3�� rs7308402R: 5��-CCTCTGCTGGGGCATATTTCAA-3�� rs2293050F: 5��-ATGGCAGACCTGTGGTGGAGAG-3�� rs2293050R: 5��-CCCTCCACCGTTTTCCTCACAC-3�� rs2139733F: 5��-GAACACCCTGACCTTAGCTGAC-3�� rs2139733R: 5��-TTTTGTTGAACCTGGGCCTCTT-3��.Amplification was done under the following condition: 95��C 2min; 11 cycles of 94��C for 20s, 65��C�C0.5��C/cycle for 40s and 72��C for 90s; 24 cycles of 94��C for 20s, 59��C for 30s and 72��C for 90s; 72��C for 2min.

Then, 10��L of PCR products was mixed with 1U SAP (Promega) and 1U Exonuclease I (Epicentre) followed by incubation at 37��C for 1h and 75��C for 15min for inactivation. The PCR products were purified. SNaPshot multiple single-base extension reaction was done with the following mixture (10��L):5��L of Brefeldin_A SNaPshot Multiplex Kit (ABI), 2��L of purified products from multiplex PCR, 1��L of primers for extension, and 2��L of ultrapure water.