A method for improving the efficiency of DNA extraction from clotted blood samples.

BACKGROUND: The efficient and rapid extraction of high-quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting-out DNA extraction methods. METHODS: Initially, 500 µL phosphate-buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18-25°C). Genomic DNA was then extracted from samples using a modified salting-out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV-visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis. RESULTS: The yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting-out method. CONCLUSIONS: Our proposed modified salting-out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

The interaction between a HSP-70 gene variant with dietary calories in determining serum markers of inflammation and cardiovascular risk.

BACKGROUND: The high prevalence of cardiovascular disease (CVD) globally is attributable to an interaction between environmental and genetic factors. Gene × diet interaction studies aim to explore how a modifiable factor interacts with genetic predispositions. Here we have explored the interaction of a heat shock protein (HSP70) gene polymorphism (+1267A > G) with dietary intake and their possible association with serum C-reactive protein (CRP), an inflammatory marker, that is a major component of CVD risk. METHODS: HSP70 genotype was determined using a TaqMan real time PCR based method.Dietary intake was assessed using a dietary questionnaire. Serum high sensitivity (Hs) CRP and other cardiovascular risk factors were assessed by routine methods. This included coronary angioplasty to determine the presence of coronary artery stenosis. RESULTS: There were significant differences between serum lipid profile and Hs-CRP across the genotypes for Hsp70. The carriers of G allele had higher serum hs-CRP concentrations, compared with the AA homozygotes, with the wild genotype. Interaction analysis showed the association was modulated by total energy intake; the interaction of high energy intake with GG genotype: RERI = 0.77, AP = 0.26, S = 1.6. CONCLUSION: We have found a significant association between the +1267A > G variant of the HSP70 gene with cardiovascular risk factors and serum hs-CRP concentrations. It is possible that a low energy diet could ameliorate the unfavorable effects of G allele of HSP70.

Serum concentrations of MCP-1 and IL-6 in combination predict the presence of coronary artery disease and mortality in subjects undergoing coronary angiography.

Cytokines play a key role in the pathogenesis of coronary artery disease (CAD). The aim of current study was to investigate the relationship between the serum concentrations of 12 cytokines with mortality and extent of CAD in individuals undergoing angiography and healthy controls. 342 CAD patients were recruited and divided into 2 groups: those with ≥50% occlusion in at least one coronary artery [Angiography (+)] or <50% obstruction in coronary arteries [Angiography (-)]. Also 120 healthy subjects were enrolled as control group. Lipid profile, fasting blood glucose, body mass index, and blood pressure were evaluated in all the subjects. An Evidence Investigator® was used for measuring 12 cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, MCP-1, IFN-γ, EGF, VEGF) using sandwich chemiluminescent assays. Univariate analysis, multivariate regression models, ROC, and Kaplan-Meier survival curves were used for exploring the candidate markers in CAD patients. Serum level of IFN-γ, IL-4, MCP-1, EGF, IL-6, and IL-8 were markedly higher in angiogram-positive patients, while VEGF concentrations were significantly (P < 0.05) lower, compared to control group. ROC analysis for MCP-1 showed that a cut-off of 61.95 pg/mL had 91% sensitivity and 91% specificity for predicting CAD patients. Moreover, >2.16 pg/mL IL-6 had a > 94% sensitivity and 70% specificity in predicting 2 years mortality in the subjects with a serum MCP-1 > 61.95 pg/ mL, and patients having IL-6/MCP-1 combination had a shorter survival.Our findings demonstrate that CAD patients with serum MCP-1 and IL-6 levels of >61.95 and >2.16 pg/mL had a higher mortality with 94.1% sensitivity and 70.5% specificity for predicting mortality in CAD patients.

Association of heat shock protein70-2 (HSP70-2) gene polymorphism with obesity.

BACKGROUND: Obesity is a major risk factor of chronic-diseases, including cardiovascular-diseases (CVD). Increasing evidence is showing the association of heat-shock protein (HSP) with type-2 diabetes and CVD; however, there is little data on the relationship between the genetic-polymorphisms of HSP70-2 with obesity. AIM: The present study has investigated the association between 1267HSP70-2 genetic polymorphism and obesity in an Iranian population with 317 subjects. SUBJECTS AND METHODS: Anthropometric parameters and biochemical measurements were measured in all the samples, while genotypes were determined using PCR-RFLP. Univariate/multivariate analyses were conducted to explore the relationship between the genetic-polymorphisms and obesity. RESULTS: The data showed a significant association between 1267HSP70-2 polymorphism in obese subjects, compared to the non-obese group. Moreover, it was observed that this polymorphism was associated with obesity in the CAD + group, which had a high BMI compared to non-obese controls. CONCLUSION: The 1267HSP70-2 polymorphism is associated with obesity in an Iranian population, supporting a possible potential genetic link between obesity and cardiovascular diseases.

Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population.

BACKGROUND: Coronary artery disease (CAD) is an inflammatory process and a major cause of mortality and morbidity. The (heat shock protein70-2) HSP70-2 gene is reported to be associated with coronary artery disease possibly by affecting the regulation of pro-inflammatory cytokines such as TNF-α. The association between CAD and the HSP70-2 gene +1267A>G polymorphism has been studied in some populations but there are no data about this association in the Iranian population. AIM: We have investigated the association between the HSP70-2 gene +1267A>G polymorphism and angiographically defined CAD within an Iranian population. METHODS: We determined the presence of the HSP70-2 gene +1267A>G polymorphism in 628 patients with CAD and 307 healthy individuals using PCR-RFLP. Of the patients, 433 (68%) had >50% stenosis (CAD+) and the remaining 195 patients had <50% stenosis (CAD-), based on coronary angiography. Angiogram positive patients were subdivided into three groups: those with single (n=113), double (n=134), and triple vessels (n=186) disease. RESULTS: A significant higher frequency of AG+GG genotypes (G allele carriers) was observed in angiogram positive and angiogram negative groups compared to controls in a dominant analysis model of the HSP70-2 gene +1267A>G position (51.2 vs. 43.2, P=0.002, OR=1.37) (51.0 vs. 43.2, P=0.01, OR=1.37). The allele frequency of the HSP70-2 G was also significantly higher in angiogram positive and angiogram negative groups compared to the control group (51.2 vs. 43.2, P=0.002, OR=1.37) (51.0 vs. 43.2, P=0.01, OR=1.37). CONCLUSION: These results suggest that HSP70-2 +1267 polymorphism may influence the risk of CAD in Iranian population, however further studies are needed to clarify the role of other HSP70-2 gene polymorphisms in the pathogenesis of the CAD.