OBJECTIVE:The aim of this study was to evaluate the roles of the Taql and Bsml vitamin D receptor gene polymorphisms in hospital mortality of burn patients.METHODS:In total, 105 consecutive burn injury patients over 18 years in age who were admitted to the Burn Unit of Bauru State Hospital from January to December 2013 were prospectively evaluated. Upon admission, patient demographic information was recorded and a blood sample was taken for biochemical analysis to identify the presence of the Taql(rs731236) and Bsml(rs1544410) polymorphisms. All of the patients were followed over their hospital stay and mortality was recorded.RESULTS:Eighteen of the patients did not sign the informed consent form, and there were technical problems with genotype analysis for 7 of the patients. Thus, 80 patients (mean age, 42.5±16.1 years) were included in the final analysis. In total, 60% of the patients were male, and 16.3% died during the hospital stay. The genotype frequencies for the Taql polymorphism were 51.25% TT, 41.25% TC and 7.50% CC; for the Bsml polymorphism, they were 51.25% GG, 42.50% GA and 6.25% AA. In logistic regression analysis, after adjustments for age, gender and total body surface burn area, there were no associations between the Taql (OR: 1.575; CI95%: 0.148-16.745; p=0.706) or Bsml (OR: 1.309; CI95%: 0.128-13.430; p=0.821) polymorphisms and mortality for the burn patients.CONCLUSIONS:Our results suggest that the Taql and Bsml vitamin D receptor gene polymorphisms are not associated with hospital mortality of burn patients.
PCR, or “polymerase chain reaction”, is one of most useful techniques in genomics research. High quality DNA extraction is necessary to obtain satisfactory results. Some methods utilized to obtain DNA from blood include a simple salting‐out procedure for extracting DNA from human nucleated cells. This method is widely used by many laboratories because it can obtain DNA with sufficient quality to realize PCR, using materials that do not pose risk to researchers and is more cost‐efficient than other approaches. Although salting‐out has spread to many laboratories, its protocols do not highlight technical details of their steps that could affect final DNA quality and, for this reason, underwent some modifications in this work in order to obtain an efficient extraction. We used 2 to 4 mL of blood for each of 30 DNA samples. For the supernatant step in first EDTA wash, we established a subtraction pattern from 15 mL to 2.5 mL, independently of initial sample volume of blood, to avoid losing leucocytes. The number of washes required is directly proportional to loss of leukocytes in the blood sample, considering that 4 mL samples can receive five washes while 2 mL samples tolerate only four. It was noted that a “centrifuge brake” can reduce DNA extraction quality, thus indicating its inadvisability. All DNA samples were submitted to spectrophotometry, which showed the DNA/Protein ratio staying between 1.1 and 1.6. According to the literature, 93% of our samples were ready for real‐time PCR after modifications, indicating that those modifications were relevant for achieving efficient extraction. Integrity was confirmed by electrophoresis. All modifications made in the salting‐out method have improved the quality and integrity of DNA samples. Grant Funding Source: FAPESP 2013/19808‐8
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