Computational protocol: Association of lincRNA-p21 Haplotype with Coronary Artery Disease in a Chinese Han Population

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Protocol publication

[…] The SNP database of lincRNA-p21 gene in the Han Chinese population was downloaded from the HapMap Project ( []. The Haploview software (version 4.2) was prerequisites for tagSNPs selection with minor allele frequency (MAF) larger than 0.05, and linkage disequilibrium (LD) patterns with r 2 > 0.8 []. Totally, four tagSNPs (rs9380586, rs4713998, rs6930083, and rs6931097) were selected for genotyping. The positions of the four tagSNPs were shown in . The r 2 information for the four tagSNPs and alleles captured accordingly was exhibited in Table S1 in Supplementary Material available online at The haplotypic blocks of the four tagSNPs were estimated by the Haploview software. Then haplotype analysis was performed through the SHEsis software ( [].Genomic DNA was genotyped using PCR-ligase detection reaction method (PCR-LDR) (Shanghai Biowing Applied Biotechnology Company). These sequences of probes and primers were shown in Table S2. The PCR reaction (20 μL) contained 50 ng genomic DNA, 0.5 μM primer mix, 1x PCR buffer, 1 U Taq polymerase, 2 mM dNTPs, and 3 mM MgCl2 on the Gene Amp PCR system 9600 (Norwalk, USA). The cycling parameters were carried out as the following procedure: 95°C for 2 min; 40 cycles at 94°C for 90 s, 56°C for 90 s, and 65°C for 30 s; and a final extension step at 65°C for 10 min. The ligation reaction for PCR product was done in a 10 μL volume containing 1 μL 1x buffer, 2 pmol probe mix, 2 U Taq DNA ligase, and 4 μL of PCR product. The LDR parameters were as the following procedure: 95°C for 2 min and 40 cycles at 94°C for 15 s and 50°C for 25 s. Then, 1 μL LDR reaction product was added into 1 μL loading buffer and 1 μL ROX. The mixture was then analyzed by the ABI Prism 3730 DNA Sequencer (Applied Biosystems, USA). For ten percent of samples, the PCR was repeated once for quality control. Repeatability of results was 100%. [...] Data analysis was performed by using the SPSS computer software (version 21). The haplotype analysis was undertaken using the SHEsis online webserver. Each tagSNP was tested in controls for confirmation with Hardy-Weinberg Expectations (HWE) by a goodness-of-fit χ 2 test. Continuous variables between the CAD/MI and control groups were presented as means ± standard deviation and compared using Student's t-test. χ 2 test was used for categorical variables in the differences of the demographic characteristics at the CAD/MI and control groups. Logistic regression analysis was used to analyze the association between the risk for CAD/MI and the tagSNPs with adjustment for risk factors of CAD (sex, age, smoking, drinking, diabetes, hyperlipidemia, and hypertension). P < 0.05 was considered to indicate statistical significance. […]

Pipeline specifications

Software tools Haploview, SPSS, SNPinfo
Applications Miscellaneous, GWAS
Diseases Coronary Artery Disease, Myocardial Infarction