Neuronal Nuclei Isolation And RNA Preparation From Mouse Brains

Human brain tissue is mainly consisted of neurons and glia cells. Neuron cell is the basic structural and functional unit of the nervous system. It has been proved that variations of neurons in cellular and molecular levels are significantly associated with nervous system diseases. There are several lines of evidences demonstrating aberrant expression of neuron specific genes is linked to nervous system diseases. However, the underlying pathogenic machnism of nervous system diseases is still unclear, it still requires further investigation. Obtaining neuron specific gene expression profiles from frozen human brain tissues will be helpful for understanding gene expression in human brain, and it will makes great contributions to pathogenesis studies of nervous system diseases. It has been proved nuclei can be perfect mRNA resoures and don’t perturb gross gene expression analysis. In our experiments, we will study on frozen human postmortem brain samples and mouse brains to isolate neuronal nuclei and extract neuron specific nuclear RNA, we will try to map neuron specific gene expression profiles through gene expression microarray or RNA-seq in the future plans.Objective: Establish the technical platform of human and mouse nuclei isolation and RNA preparation.Methods:1. Lysate the brain tissue samples in cell lysis buffer to make nuclei suspension.2. Use sucrose density gradient centrifugation to isolate pure nuclei.3. Use fluorochrome conjugated NeuN antibody to specifically label the nuclei and obtain purified neuronal nuclei through fluorescence active cell sorting.4. Centrifuge and collect the sorted nuclei, use Trizol or RNA extraction kit to isolate neuronal nuclear RNA.5. Analyze RNA quality by Agilent2100bioanalyzer.Results:1. We can isolate purified neuronal nuclei from human and mouse brain tissues though fluorescence active cell sorting, nuclei can keep intact morphology throughout the experiments.2. We can isolate nuclear RNA from the sorted neuronal nuclei.3. We can obtain the neuron proportion data of human brain samples, and find neuron proportion are different among different brain regions and different individuals.4. RNA2100analysis results demonstrate RIN value of all fresh mouse brain tissue RNA are over7, but relative low in frozen human brain tissue RNA. All nuclear RNA have very low RIN value. These results imply that RNA2100analysis is not suitable for nuclear RNA quality analysis.Conclusion:1. We successfully established the platform of human and mouse nuclei isolation and RNA preparation.2. Current RNA2100analysis system is not suitable for nuclear RNA quality analysis.3. Neuron proportions are different among different cerebral cortex regions.