Analysis of RNA by Flow Cytometry (CAT#: STEM-CBT-0064-WXH)

1.The SmartFlareTM (EMD Millipore) system utilizes target specific probes which are gold nanoparticles conjugated to oligonucleotides and are taken up by live cells through endocytosis. The probes are designed such that once inside the cell if the probe binds to the mRNA of interest, fluorescence is emitted and can be detected by a flow cytometer.
2.The PrimeFlowTM (ThermoFisher Scientific) approach uses fluorescent in situ hybridization that allows specific hybridization of oligonucleotide probes to specific regions across the target mRNA sequence in cells. Then, using the branched DNA technology, the signal is amplified through a series of sequential hybridization steps to form a tree-like structure. Pre-amplifier molecules hybridize to their respective pair of bound oligonucleotide probes (the trunk of the tree), to which hybridizes the amplifier molecules (forming the branches). Finally, multiple label probes hybridize to the amplifiers (the leaves of the tree). Fluorescence can then be detected using a standard flow cytomete

• Probe mRNA when an antibody to the protein target is unavailable.
• Analyze mRNA expression at the single-cell level.
• Compare RNA and protein kinetics in the same cell.
• Detect microRNA (miRNA).
• Evaluate viral RNA in infected cells.
• Validate single-cell RNA sequencing results.

1.Label proteins with antibody(optional)
2.fix and permeabilize cells
3.Label intracellular proteins with antibodies(optional)
4.Incubate cells with gene-specific probe sets
5.Hybridize with preamplifier and amplifier DNA
6.Add fluorescently labeled probes to cells
7.Process cells using a flow cytometer