Chemical Contaminants Inhibit Probe Hybridization (RNA Depletion for RNA-seq)

Question

Accepted Answer

Depletion efficiency is reduced or completely absent. Target RNA remains at high levels after treatment despite using validated probe designs. Common causes: 1) Salt contamination (Mg2+ or guanidinium salts) interferes with probe-target hybridization 2) Organic solvent contamination (phenol or ethanol) disrupts hybridization chemistry 3) RNA sample not properly resuspended in nuclease-free water 4) Carryover contaminants from RNA extraction or purification steps Solutions: 1) Ensure RNA samples are free of salts (e.g., Mg2+, guanidinium salts) before depletion 2) Remove all traces of organic solvents (phenol and ethanol) through proper RNA purification 3) Resuspend purified RNA in nuclease-free water only 4) Perform additional ethanol wash steps or column purification if contamination is suspected 5) Verify RNA quality using spectrophotometry (260/230 ratio >1.8) to assess salt/organic contamination