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Microbiology JoVE (Open Access) Citable · DOI

MISSION esiRNA for RNAi Screening in Mammalian Cells

DOI: 10.3791/2008-v
What you'll learn
  • ✓ Design and optimize esiRNA transfection protocols for mammalian cells
  • ✓ Execute a primary RNAi screen targeting cell cycle genes
  • ✓ Validate hits using secondary screening and data analysis methods
Protocol

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Difficulty
intermediate
Total time
~5–7 days (optimization 1–2 days, primary screen 2–3 days, secondary validation 2–3 days)
Model organism
Mammalian cultured cells (human-derived)
Biosafety
BSL-1

Steps

1
Select and prepare MISSION esiRNA libraries

Review high-quality esiRNA library characteristics and storage protocols. Understand esiRNA design principles for robust RNAi knockdown in mammalian systems.

▶ 02:22
2
Optimize transfection conditions for target cells

Establish optimal transfection reagent concentration, timing, and cell confluency to maximize esiRNA delivery efficiency prior to screening.

▶ 03:49
3
Set up assay and prepare primary screen

Prepare cell culture plates, transfection templates, and phenotypic readouts (e.g., viability or proliferation assays) for high-throughput screening.

▶ 05:28
4
Execute primary RNAi screen on target gene pool

Perform large-scale transfection of esiRNA library targeting cell cycle genes and measure phenotypic outputs to identify candidate hits.

▶ 09:34
5
Conduct secondary validation screen on hits

Re-test primary screen candidates with individual esiRNAs to confirm specificity and eliminate false positives.

▶ 12:03
6
Analyze and interpret screening results

Apply statistical methods and data visualization to identified hits from mammalian cell cycle screening and present representative outcomes.

▶ 13:40

🚨 Failure Case Library (3) + Submit your own case

severe
Empty/negative control also shows fluorescence (high false-positive background)
Untransfected or empty-vector wells show a clearly green/red signal — you can't tell if the target plasmid is really expressing.
💡 4 · ✓ 4
severe
siRNA: qPCR shows knockdown but Western blot shows no change
qPCR confirms target mRNA is significantly reduced, but the corresponding protein band on Western blot looks unchanged.
💡 5 · ✓ 4
moderate
Very high replicate-well variation (poor reproducibility)
Parallel wells show very different fluorescence intensity / positive-cell rates; data fluctuates wildly between replicates.
💡 4 · ✓ 4
💬 Comments coming soon