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Detection of mRNA expression in fetal brain regions using LCM and RT-PCR

Introduction. Mechanisms that regulate the development of different brain regions is not fully understood Examining expression pattern of different mRNA species in discrete brain regions in developing brains may help understand the developmental process . Project Purpose. To develop a method to Amplify cDNA, representing different mRNA species, from laser captured cells of rat fetal brains.

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Detection of mRNA expression in fetal brain regions using LCM and RT-PCR

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    1. Detection of mRNA expression in fetal brain regions using LCM and RT-PCR Brian McMaster Neuroscience Scholar 2008 The Center for Psychiatric Neuroscience, UMMC Mentor Ujjwal K. Rout, M.Phil., Ph.D. Division of Pediatric Surgery Research Labs, UMMC, Jackson, MS

    3. Project Purpose To develop a method to Amplify cDNA, representing different mRNA species, from laser captured cells of rat fetal brains

    4. Methods Cryo-preservation of rat fetal heads Fetal Rat brain sectioning Staining slides with HistoGene kit (Arcturus) LCM and collection of cells from discrete brain regions Isolation of total RNA from collected tissue samples RT-PCR amplification of cDNA of interests

    5. Methods Fetal heads were cryo-preserved and stored at (-80oC) Frozen Heads were mounted with OCM medium on dry ice 20 micron thick sections were cut using a Cryostat Sections were loaded onto RNase free glass slides Sections were preserved at -80oC

    6. Methods Brain sections were stained using an Arctus Kit

    7. Methods Capturing cells from discrete brain regions using LCM system

    8. Methods Isolation of total RNA from captured cells

    9. Methods PCR amplification of cDNA species synthesized from LCM captured cells

    10. Results

    11. Results

    12. Results PCR amplifications of cDNA species synthesized from the LCM captured cells

    13. Conclusions and Future Directions Methods for the isolation of cells from specific fetal brain regions and detection of mRNA of interests were successfully completed. Use Real-time PCR techniques to study the pattern of gene expression in different brain regions of rat fetal brains. Use information on the relative abundance of mRNA of interests in different fetal brain regions to understand their involvement in the brain development.

    14. Acknowledgments Dr. Rout Department of Surgery Center for Psychiatric Neurosciences

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