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 wide spread application to and development for flow cytometry. Two new techniques, PrimeFlowTM (Affymetrix/eBioscience) and SmartFlareTM (EMD Millipore) have recently been commercially introduced and are seeing increasing use among research scientists.
This tutorial will discuss:
- The methodology leading up to the current techniques
- The PrimeFlowTM branched DNA methodology and applications
- The SmartFlareTM technique and procedures
- Pitfalls, quality control and analysis strategies for both Techniques
- Methods to simultaneously combine mRNA measurements with labeling of surface and intracellular proteins
The PrimeFlowTM RNA assay system from Affymetrix/eBioscience 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 branched DNA signal amplification. Protocols and data will be presented showing the usefulness of this system to identify RNA expression on bone marrow B cells subsets. We will show a direct comparison of this method to a much more time consuming process of sorting cells for microarray analysis. This method provides a sophisticated molecular tool for the characterization of gene expression on rare cell subsets within tissue.
The SmartFlareTM system from EMD Millipore 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 this system to sort cells based on mRNA expression, for several applications such as isolation of cancer stem cells from tumors and optimizing shRNA or CRISPR knockdown clone selection. This new technology now enables the non-destructive detection of intracellular epitopes in living cells.
By the end of this tutorial, attendees will 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.
Course Details or Outline:
1. Overview of methods to detect RNA in bulk preparations of cells
2. Initial flow cytometric methods to detect HIV by in situ PCR
3. RNA-Seq and Fluidigm methodogies
B. PrimeFlow - branched DNA
2. Method and data analysis
4. Comparison of flow to microarray based expression profiling
1. Theory of detecting RNA in live cells
2. Protocols and data analysis
4. Use for isolation of cancer stem cells from tumors
5. Optimizing shRNA or CRISPR knockdown clone selection