Considerations of Gene Expression Analysis Methods for Gapmer Antisense Oligonucleotide-Treated Samples

Probe-based PCR assays are commonly used to evaluate gene expression in antisense oligonucleotide (ASO)-treated samples, but assay design and postextraction processing may introduce substantial measurement bias. We investigated workflow-dependent differences in measured knockdown across PCR-based and RNA-sequencing assays using an allele-specific gapmer ASO, targeting a linked single nucleotide polymorphism of a dominant-negative SPTAN1 variant. Exogenous ribonuclease H (RNase H) activity during reverse transcription or ribosomal RNA (rRNA)-depletion inflated apparent knockdown and reduced RNA-sequencing coverage at the ASO-binding site. In droplet digital PCR (ddPCR), allele-specific detection probes overlapping the ASO-binding site produced substantially greater apparent knockdown than nonoverlapping probes, particularly following workflows with active RNase H. Spike-in experiments showed that this bias was not reproduced by adding ASO to purified RNA before complementary DNA (cDNA) synthesis or to cDNA before ddPCR, but emerged when ASO was present during rRNA depletion. Together, these findings show that exogenous RNase H activity during RNA processing and ASO–probe overlap can distort measurements of gapmer-mediated transcript reduction. Poly(A)-based RNA sequencing, reverse transcriptase with reduced RNase H activity, and nonoverlapping PCR detection probes can minimize measurement bias when evaluating gapmer ASO-mediated knockdown.