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Understanding miRNA Turnover: A Study of miRNA Half-Life

Understanding miRNA Turnover: A Study of miRNA Half-Life. Dominic McDonald Massachusetts Institute of Technology Mentor: Jun Lu, Ph. D Todd Golub Laboratory Broad Institute of MIT and Harvard. What are miRNAs?. miRNAs are short non-coding RNAs

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Understanding miRNA Turnover: A Study of miRNA Half-Life

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  1. Understanding miRNA Turnover: A Study of miRNA Half-Life Dominic McDonald Massachusetts Institute of Technology Mentor: Jun Lu, Ph. D Todd Golub Laboratory Broad Institute of MIT and Harvard

  2. What are miRNAs? • miRNAs are short non-coding RNAs • Post-transcriptional regulators of gene expression • miRNAs regulate diverse biological and disease processes

  3. miRNA Biogenesis Zamore PD & Haley B. Science 2005

  4. How do miRNAs turnover?

  5. Approach to Measure miRNA t1/2 Culture Cells in Triplicates Treat Cells with Actinomycin D Harvest RNA at 6 time points throughout the day Measure miRNA and control gene expression levels using Luminex NextGen and RT-PCR

  6. Maintained 26 different cancer cell lines. Due to time constraints only able to collect data from 9 different cancer cell lines: HD-MY-Z, 697, HL60, K562, Ramos (RA1), RS4;11, SEMK2, U937, and HEL cell lines. Triplicates of every cell line done at 6 time points each for a total of 162 samples. Cell Lines

  7. Control Gene Degradation c-myb

  8. Control Gene Degradation c-myb

  9. Control Gene Degradation c-myc

  10. MicroRNA Profiling Lu, J Nature 2005

  11. RT-PCR Validation • Use additional methodology to validate results • Use RT-PCR for validation • Measure miR-125a in K562 cells • Measure u6 small RNA as a control

  12. RT-PCR Validation

  13. RT-PCR Validation

  14. RT-PCR Validation

  15. What Does This All Mean? • Most miRNAs have long t1/2 (over 24 hours) • We have not observed any fast degrading miRNAs (t1/2 less than 8 hours)

  16. Why would nature create a class of stable cellular regulators?

  17. Our Team: Jun Lu Hao Zhang Judy Wang Golub Lab: Todd Golub Jinyan Du Broad Institute: Shawna Young Bruce Birren Lucia Vielma Maura Silverstein Acknowledgements

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