Home › Cell Biology › Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
Cell Biology JoVE (Open Access) Citable · DOI

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

DOI: 10.3791/51420-v
What you'll learn
  • ✓ Perform click-chemistry RNA labeling under hypoxic conditions
  • ✓ Visualize single-cell RNA synthesis changes using fluorescence microscopy
  • ✓ Analyze cell-to-cell heterogeneity in global RNA expression
Protocol

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Difficulty
advanced
Total time
~4–6 hours per sample batch (culture treatment + assay + imaging)
Model organism
Cell culture (mammalian; specific line not specified in abstract)
Biosafety
BSL-1

Steps

1
Culture and expose cells to hypoxia

Prepare cultured cells and apply hypoxic treatment conditions. Maintain cells under controlled oxygen levels for the specified duration.

▶ 02:14
2
Perform click-iT RNA labeling assay

Apply click-chemistry reagents to label newly synthesized RNA in fixed or live cells under hypoxic conditions. Follow manufacturer protocol for incubation and washing steps.

▶ 04:58
3
Acquire and analyze fluorescence microscopy images

Image labeled RNA using fluorescence microscopy and perform quantitative image analysis to measure single-cell RNA synthesis levels. Extract intensity and distribution metrics per cell.

▶ 09:59
4
Evaluate RNA expression in hypoxic cells

Compare global RNA synthesis between hypoxic and normoxic control cells, focusing on cell-to-cell heterogeneity and population-level trends.

▶ 12:41
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