Flow cytometric analysis of double-stranded RNA content distributions

Area of Science:

• Cell Biology
• Biochemistry
• Biotechnology

Background:

• Accurate quantification of cellular RNA content is crucial for understanding cell proliferation and disease states.
• Flow cytometry (FCM) offers a high-throughput method for cellular analysis.
• Distinguishing between normal and cancerous cells based on molecular content is a key challenge in diagnostics.

Purpose of the Study:

• To develop and validate a novel staining procedure for quantitative analysis of cellular RNA content using FCM.
• To investigate the relationship between double-stranded RNA (dsRNA) content and cell proliferation.
• To differentiate between tumor cells and normal cells based on dsRNA levels.

Main Methods:

• Cells were fixed in ethanol and treated with DNAse.
• Staining was performed using propidium iodide, a fluorochrome that binds to dsRNA.
• Quantitative analysis of dsRNA content distributions was conducted using FCM.

Main Results:

• FCM analysis confirmed that over 90% of fluorescence originated from dsRNA-bound fluorochrome.
• Maximal dsRNA content correlated with periods of active cell proliferation (S-phase index) in HeLa S3, L1210 leukemia, and regenerating bone marrow cells.
• Distinct dsRNA fluorescence peaks differentiated L1210 tumor cells from non-tumor cells and showed higher dsRNA in lymphoma cells compared to normal thymic cells.

Conclusions:

• The described staining procedure provides a reliable method for quantitative dsRNA analysis by FCM.
• Cellular dsRNA content is a valuable indicator of cell proliferation status.
• This FCM-based dsRNA quantification can aid in distinguishing tumor cells from normal cells.