Project description:Diabetes exerts adverse effects on the initiation or progression of diabetes and metabolic syndrome of in the next generation. In past studies, limited attention has been given to the fathers’ role in shaping the metabolic landscape of offspring. Our study was designed to investigate how paternal hyperglycemia exerts an intergenerational effect in mammals as well as the underlying mechanisms. Hyperglycemia was introduced in male rats by intraperitoneally injected streptozotocin and these males were bred with healthy female rats to generate offspring. The metabolic profiles of the progeny were assessed, and DNA methylation profiles as well as gene expression related to lipid metabolism were investigated.
Project description:DNA methylation is an epigenetic mark that has a crucial role in regulating gene expression. Aberrant DNA methylation results in severe diseases in humans, such as cancer, autoimmune disease, atherosclerosis, and cardiovascular diseases. Whole-genome bisulfite sequencing and methylated DNA immunoprecipitation are available to study DNA methylation changes, but they are typically used on a few samples at a time. Here, we developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), that can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis.
Project description:We applied small RNA Solexa sequencing technology to identify microRNA expression in human liver samples from surgically removed liver tissues including three normal liver tissues (distal normal liver tissue of liver hemangioma), an hepatitis B virus (HBV)-infected liver, a severe chronic hepatitis B liver, two HBV-related hepatocellular carcinoma (HCC), an hepatitis C virus (HCV)-related HCC, and an HCC without HBV or HCV infection. All samples were collected with the informed consent of the patients and the experiments were approved by the ethics committee of Second Military Medical University, Shanghai, China. We investigated the miRNome in human normal liver and suggested some deregulated abundantly expressed microRNAs in HCC. center_name: National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China.
Project description:Subcellular fractionation of rat liver from control or Triton WR-1339-treated animals fasted overnight. Density based separation of a differential centrifugation fraction enriched in lysosomes and peroxisomes. Samples were digested with trypsin, labeled using TMT6, pooled, and further fractionated as described in methods and protocols.
Project description:Subcellular fractionation of rat liver from control or Triton WR-1339-treated animals fed ad libitum. Density based separation of a differential centrifugation fraction enriched in lysosomes and peroxisomes. Samples were digested with trypsin, labeled usiniTAQ8, pooled, and further fractionated as described in methods and protocols.