Probe Integrity Compromised During Storage or Synthesis

Symptom

Target RNA depletion efficiency is significantly reduced or absent. Sequencing shows minimal reduction in targeted reads despite proper protocol execution.

1. 1 Probes degraded due to improper storage conditions or handling
2. 2 Poor quality oligo synthesis from supplier with length distribution outside 40-60 nt range
3. 3 DNase contamination in input RNA degraded single-stranded DNA probes during hybridization

1. 1 Order probes from trusted oligo synthesis provider and store according to specifications
2. 2 Evaluate probe pool using single-stranded DNA size estimation method to confirm length distribution is 40-60 nt
3. 3 Purify RNA completely from DNase I enzyme after treatment - residual DNase I activity will degrade probes
4. 4 Ensure any residual DNase I is removed from sample before adding depletion probes