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MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow

  • Describing tools developed to isolate total RNA, for mRNA and miRNA analyses, in the context of epigenetic workflows with cancer cell lines
  • Highlight two projects that combine novel real-time cell health assays (viability and cytotoxicity) with total RNA purification in the same cells in multiple culture formats, with downstream analysis of miRNA effects

Summary

The requirements for nucleic acid purification for use in RNA profiling have expanded with the growing interest in the role of microRNAs (miRNAs) and other small non-coding RNAs in cancer cell growth and metastasis.  This evolution of expression analysis highlights the need for more sophisticated tools for total RNA isolation beyond traditional mRNAs. Here we describe tools we have developed to isolate total RNA, for mRNA and miRNA analyses, in the context of epigenetic workflows with cancer cell lines.  In this presentation, we highlight two projects that combine novel real-time cell health assays (viability and cytotoxicity) with total RNA purification in the same cells in multiple culture formats, with downstream analysis of miRNA effects. 

In the first study, cells in both 3D and standard culture formats are dosed with a common chemotherapeutic then analyzed in real-time for cell viability and cytotoxicity.  After determining potency of the drug, total RNA isolated with a novel extraction kit and miRNAs analyzed for expression changes using RT-qPCR.

In the second study, two breast cancer cell lines, with distinct responses to histone de-acetylase (HDAC) inhibitors, are analyzed for viability, cytotoxicity, HDAC activity, and mRNA plus miRNA expression profiling in a single experiment.


Speaker

brad-hook-125x125

Brad Hook, PhD
Senior Applications Scientist

Brad Hook is a Senior Application Scientist at Promega Corporation. Scientific Applications Services drives revenue growth through scientific support of commercial sales. As an Application Scientist, Brad advises, tests and adapts existing products to meet specific customer needs. He facilitates adoption of products by performing demonstrations, troubleshooting experiments, and giving seminars. 

Prior to joining Promega, Brad received his Ph.D. in Biochemistry from the University of Wisconsin-Madison where he studied developmental biology with a focus on RNA regulation.  Brad joined Promega R&D in 2007 and contributed to the development of HaloTag based products.  Brad joined the Applications Group in 2010 and has since written many articles highlighting applications in the Proteomics, Genomics, Genetic Identity, Applied Markets and Cellular Analysis product areas.

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