Probe Integrity Compromised During Storage or Synthesis (RNA Depletion for RNA-seq)

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Target RNA depletion efficiency is significantly reduced or absent. Sequencing shows minimal reduction in targeted reads despite proper protocol execution. Common causes: 1) Probes degraded due to improper storage conditions or handling 2) Poor quality oligo synthesis from supplier with length distribution outside 40-60 nt range 3) DNase contamination in input RNA degraded single-stranded DNA probes during hybridization Solutions: 1) Order probes from trusted oligo synthesis provider and store according to specifications 2) Evaluate probe pool using single-stranded DNA size estimation method to confirm length distribution is 40-60 nt 3) Purify RNA completely from DNase I enzyme after treatment - residual DNase I activity will degrade probes 4) Ensure any residual DNase I is removed from sample before adding depletion probes