RNA Flow Cytometry

About the Presenters

Steve McClellan, BS, MT(ASCP)
Manager of Basic & Translational Research Operations
University of South Alabama Mitchell Cancer Institute

Steve McClellan, BS, MT(ASCP) is a life science professional with over 25 years of expertise in advanced flow cytometry and cell sorting, as well as basic and clinical research in the areas of cancer biology, stem cell therapy, transplant immunology, and xenotransplantation. He is currently manager of basic and translational research operations at the University of South Alabama Mitchell Cancer Institute, where he also serves as chief of flow cytometry and imaging core laboratories. For the past seven years, his lab has been conducting research on cancer stem cells, specifically developing better methods of purification, culture, and analysis at both genetic and functional levels, as well as using cancer stem cells in HTS drug discovery.

Paul K. Wallace, PhD
Director of the Flow and Image Cytometry Department
Roswell Park Cancer Institute

Paul K. Wallace, PhD has served since 2003 as director of the Flow and Image Cytometry Department and is professor of oncology at Roswell Park Cancer Institute (RPCI) in Buffalo, NY. He is also associate professor of microbiology & immunology at Dartmouth College and associate professor of biotechnical and clinical laboratory sciences at the University of Buffalo. He is past president of ISAC and a councilor of the International Clinical Cytometry Society. Under his direction, the Flow and Image Cytometry Department at RPCI offers a strong combination of clinical and research missions. The department’s clinical emphasis is on the diagnosis and monitoring of patients with leukemia and lymphoma. In addition, it serves as a core reference facility performing immunophenotyping and immune monitoring studies on samples from patients enrolled in clinical trials for several biotech and grant-funded organizations. The department’s research focus is on myeloid cell biology and translational research utilizing flow cytometry.

Webinar Summary

Flow cytometry permits the simultaneous measurements of many biomarkers in individual cells from bulk populations. Until now analysis has been limited, however, to primarily analysis of proteins and total DNA or highly abundant DNA sequences. Since most RNA gene transcripts are present at very low quantities, our ability to detect these mRNA species by flow cytometry has been limited. In 1993 Patterson et. al. (Science, 1993. 260:976) used a PCR driven in situ hybridization technique to detect HIV mRNA in infected cells; however, the stringent conditions required by this technique prevented its widespread application to and development for flow cytometry. Two new techniques, PrimeFlowTM (Affymetrix/eBioscience) and SmartFlareTM (EMD Millipore) have recently been commercially introduced and are increasingly used among research scientists.

The PrimeFlow RNA assay system allows for the determination of differential RNA expression within a mixed population of cells. The assay improves sensitivity and lowers background of fluorescent in situ hybridization (FISH) through the use of a branched DNA signal amplification. In contrast, the SmartFlareTM system employs a gold nanoparticle that is actively endocytosed into most cell types. In the cytoplasm, interaction with the specific mRNA target causes the generation of a fluorescent signal, which can be detected by flow cytometry or microscopy. Protocols and data will be presented showing the usefulness of both systems. By the end of the webinar, participants should have an understanding of which technique is most appropriate for their applications; how to apply them to their research; and most importantly, gain an understanding of the pitfalls and solutions from individuals who have learned these the hard way.

CMLE Credit: 1.0