Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.

Objective:This study was to investigate the expression of long noncoding RNA (lncRNA) KCNQ1OT1 during the differentiation program of human preadipocyte and to look for the changes of its expression in adipose tissue in obese subject, as well as to clarify the correlation between KCNQ1OT1 and obesity, and to provide clues for further understanding the role of lncRNA in the development of obesity.Methods:Quantitative PCR was used to detect the expression of KCNQ1OT1 in human preadipocyte at day 0, 1, 3, 5, 9, and 12 during differentiation program, quantitative PCR was also used to detect KCNQ1OT1 expression in white adipose tissue of obese and normal people, which related to PPIA internal reference gene. Pearson correlation analyses were used to explore the relationships of KCNQ1OT1 with body mass index, triglyceride, and total cholesterol.Results:During differentiation program, the relative expression of KCNQ1OT1 levels at day of 1, 3, 5, 9, and 12 were (25.89±3.10), (24.78±5.58), (15.53±2.11), (6.75±0.71), (4.81±0.84), which showed an upward trend compared with day 0. This difference was significant ( P<0.01), especially in the early stage of differentiation (day 1 and day 3). The relative expression of KCNQ1OT1 in visceral adipose tissue of obese subjects was 0.79±0.05, which was significantly higher than that of normal people ( P<0.01). KCNQ1OT1 was positively correlated with body mass index ( r=0.569, P<0.01), triacylglycerol content ( r=0.489, P<0.05), and total cholesterol content( r=0.591, P<0.01). Conclusion:KCNQ1OT1 expression level, which was up-regulated in adipose tissue from obese subjects, increased during the differentiation program of preadipocytes, and also positively correlated with body mass index and serum triglyceride content. These results suggest that KCNQ1OT1 may be an important regulator of human preadipocyte differentiation and a potential target for prevention and treatment of obesity.