Using intracellular SCGB1A1-sorted, formalin-fixed club cells for successful transcriptomic analysis

Area of Science:

• Cell biology
• Molecular biology
• Respiratory medicine

Background:

• Intracellular markers are crucial for identifying specific cell types, but intracellular staining for cell sorting is challenging.
• Fixation, permeabilization, and RNase activity can degrade mRNA quality, hindering transcriptomic analysis.
• Optimizing protocols for intracellular staining is essential for preserving RNA integrity in sorted cells.

Purpose of the Study:

• To develop and optimize a protocol for intracellular staining and cell sorting of human bronchial club cells (SCGB1A1+).
• To ensure the preservation of high-quality RNA suitable for microarray analysis after intracellular staining and cell sorting.
• To generate the transcriptomic signature of club cells using the optimized protocol.

Main Methods:

• Differentiated human bronchial epithelial cells from air-liquid interface cultures were used.
• Cells were stained with intracellular markers for club cells (SCGB1A1+) and sorted using flow cytometry.
• A modified fixation and permeabilization protocol (4% paraformaldehyde, 0.1% Triton/0.1% saponin) was employed, including RNase inhibitors.

Main Results:

• The optimized protocol successfully stained and sorted SCGB1A1+ club cells using intracellular markers.
• RNA extracted from sorted cells was of sufficient quality for microarray analysis, as confirmed by Bioanalyzer.
• The addition of RNase inhibitors significantly improved RNA quality, enabling transcriptomic profiling.

Conclusions:

• A robust protocol for intracellular staining and cell sorting of human bronchial club cells was established.
• This method allows for the generation of transcriptomic signatures from specifically sorted club cells.
• The protocol overcomes challenges in preserving RNA quality during intracellular staining for cell sorting.