DEC 16, 2014 08:00 AM PST

Detection of RNAs with flow cytometry

Speakers
  • Director of International Programs at the Ragon Institute, MGH, MIT, and Harvard
    Biography
      Dr. Filippos Porichis is the Director of International Programs at the Ragon Institute and serves as a member of the Executive Committee of the MGH Center for Global Health. He is an Assistant in Immunology at Massachusetts General Hospital and an Instructor of Medicine at Harvard Medical School. His research group at the Ragon Institute focuses on the immunological aspects of T cell function in HIV and Tuberculosis (TB) infections, and the implementations of novel techniques in order to evaluate the immunoregulatory networks that lead to T cell dysfunction.

    Abstract:

     

    Single-cell analysis is one of the most rapidly growing fields in biomedical research that is significantly expanding our understanding on the biologic characteristics of various diseases. Flow cytometry has been the gold standard for immunologists due to its high throughput characterization of various cell types, however availability of antibodies can be restrictive. Recent advances in molecular biology enabled techniques to perform analysis of transcriptional expression at the single-cell level but these techniques follow laborious methods that involve cell sorting of individual cells and perplexing protocols for the isolation and amplification of RNA.

    This webinar will describe a novel flow-FISH (Fluorescent in situ hybridization) method for high-throughput detection of mRNA and miRNA at the single-cell level with flow cytometry. It is based on a branched DNA technology that allows robust (up to 8,000 fold) signal amplification through a sequential hybridization of DNA branches followed by staining with labelled probes. By investigating human blood samples, we show that this novel technique enables simultaneous detection of several mRNA molecules in various leukocyte subsets identified by antibody staining for cell surface markers. Similarly, we show how this technique can be readily used for detection of small miRNA molecules that play major regulatory roles in eukaryotic cells without being translated to proteins. Finally, we prove that this technique can be used in combination with the ImageStream technology, allowing for high-throughput visualization of mRNA in combination with protein expression at the single cell level. In conclusion, we describe a user friendly protocol that enables immunologists to perform in depth transcriptional analysis of single cells using flow cytometry.


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