Chemical Contaminants Inhibit Probe Hybridization

Symptom

Depletion efficiency is reduced or completely absent. Target RNA remains at high levels after treatment despite using validated probe designs.

1. 1 Salt contamination (Mg2+ or guanidinium salts) interferes with probe-target hybridization
2. 2 Organic solvent contamination (phenol or ethanol) disrupts hybridization chemistry
3. 3 RNA sample not properly resuspended in nuclease-free water
4. 4 Carryover contaminants from RNA extraction or purification steps

1. 1 Ensure RNA samples are free of salts (e.g., Mg2+, guanidinium salts) before depletion
2. 2 Remove all traces of organic solvents (phenol and ethanol) through proper RNA purification
3. 3 Resuspend purified RNA in nuclease-free water only
4. 4 Perform additional ethanol wash steps or column purification if contamination is suspected
5. 5 Verify RNA quality using spectrophotometry (260/230 ratio >1.8) to assess salt/organic contamination