Home › Cell Biology › Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection
Cell Biology JoVE (Open Access) Citable · DOI

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

DOI: 10.3791/3655-v
What you'll learn
  • ✓ Design and validate locked nucleic acid probes for bacterial small RNA detection.
  • ✓ Perform fixation, permeabilization, and hybridization on bacterial cells.
  • ✓ Acquire and interpret flow cytometry-FISH data from single bacterial cells.
  • ✓ Distinguish specific small RNA expression patterns using fluorescence readouts.
Protocol

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Difficulty
advanced
Total time
~4–6 hours per sample (fixation through flow cytometry acquisition)
Biosafety
BSL-1

Steps

1
Design LNA probes and plan experimental strategy

Select and design locked nucleic acid probes targeting bacterial small RNA or mRNA sequences. Plan probe labeling, concentration, and positive/negative controls for flow cytometry.

▶ 01:25
2
Fix bacterial cells with crosslinking agent

Treat cultured bacterial cells with fixative to preserve RNA and cellular morphology while preparing for downstream hybridization steps.

▶ 03:36
3
Treat with DEPC and permeabilize cell membranes

Apply diethylpyrocarbonate (DEPC) treatment to inactivate RNases, then permeabilize bacterial cell walls to allow probe entry.

▶ 03:36
4
Hybridize LNA probes and perform post-hybridization washes

Incubate fixed, permeabilized cells with fluorescently labeled LNA probes under stringent conditions, then wash to remove unbound probe.

▶ 04:53
5
Block non-specific binding and apply counterstaining

Apply blocking buffer to reduce background fluorescence, then add nucleic acid counterstains or additional markers for flow cytometry gating.

▶ 05:47
6
Assess cell aggregation and collect flow cytometry data

Evaluate cell singlets versus aggregates by flow cytometry, then acquire fluorescence intensity data to quantify small RNA expression per cell.

▶ 07:59
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