Flow Cytometry Applications for Gene Expression, RNA Detection, and Cell Analysis

Author: Mike Blundell | Reviewer: Chloe Fenton

What Can Flow Cytometry Be Used for Beyond Core Applications?
In addition to core analyses such as immunophenotyping and cell cycle studies, flow cytometry can also be used for functional and molecular applications, including gene expression analysis, absolute cell quantification, particle internalization assays, and RNA detection.

Expanded Flow Cytometry Applications Overview

Flow cytometry can be applied to a range of specialized assays that extend beyond standard phenotyping and proliferation workflows. These applications enable measurement of gene expression, molecular activity, and functional cellular processes at the single-cell level.

Applications of flow cytometry and their uses.

Gene Expression and Transfection

Fluorescent proteins to determine gene expression and transfection efficiency in both live and fixed cells are widely used in flow cytometry, and are particularly useful when performing cell sorting experiments. They can be used as reporters of transcription factors, promoter activity, and cellular expression patterns, as well as screening for RNAi and CRISPR activity due to the high-throughput capacity of flow cytometry. Fluorescent proteins can also be photoactivatable, photoswitchable, and used in FRET experiments. Initially only green fluorescent protein (GFP), there are now over a hundred fluorescent proteins that excite and emit at various wavelengths, making them perfect for multicolor flow cytometry.

Absolute Quantification

Although flow cytometry can quantify expression of markers both on and in cells, they do not provide information on the cell concentration or necessarily absolute quantification. To overcome this, fluorescent beads can be added and counted. If a known amount of beads at a known concentration is added to your sample and acquired, the number of beads will be relative to the number of cells. Some cytometers can give accurate cell counts by measuring the volume of sample acquired and, in this case, the number of cells per mL can be measured.

Particle Internalization

Internalization of particles, cell surface markers, and antigens can occur through various cellular processes, e.g., phagocytosis. Flow cytometry has proved to be an effective method of quantifying this through fluorescently labeling the particle that is to be internalized. By utilizing dyes that alter their fluorescent characteristics when internalized or by quenching surface bound fluorescence, the difference between surface and internalized particles can be measured.

Fluorescence In Situ Hybridization and RNA Detection

Fluorescence in situ hybridization (FISH) was first performed in flow cytometry in the late 1990s to determine telomere length. Fluorescent nucleic acid probes were used to highlight specific repeat sequences and then fluorescence measured using specific software. Since then, RNA expression protocols have been developed that allow quantification of the levels of mRNA. This is a powerful tool as it can be performed in combination with surface staining to identify specific cells and subsets, whereas quantitative RT-PCR, while very sensitive, will only give information on a cell population.

Choosing the Right Flow Cytometry Application

• Gene expression assays—ideal for studying transcriptional activity and transfection efficiency
• Absolute quantification—used when accurate cell counts are required
• Internalization assays—suited to analyzing particle or antigen uptake
• RNA detection—enables measurement of mRNA within specific cell populations

For a broader overview of key techniques and use cases, explore our full range of flow cytometry applications.