Analytical methods for oligonucleotides or RNAs

qPCR (amplification)Unmodified oligonucleotides (chemical modifications may affect the assay)High sensitivity, broad dynamic range (4–8 orders of magnitudes), medium to high throughputExtensive sample preparation
Depends on primers and fluorophore
Low resolution, accuracy, and precision
Limited specificity and potential matrix effects
May not distinguish metabolites from the analyte
Hybridization-based assays or ELISA (no amplification)Modified or unmodified oligonucleotidesHigh specificity (sequence dependent)Depends on the reliability of probe, detection marker, or antibody
Wide dynamic rangePossible matrix effects
Good sensitivityMay not distinguish metabolites from the analyte
Reasonable accuracy and reproducibility
LC-UV (or FL)Modified or unmodified oligonucleotidesHigh specificity after separationExtensive sample preparation
Good accuracyLow sensitivity
Broad dynamic rangeRequires good separation
Low to medium throughput(Sensitive FL detection depends on the fluorophore-labeled probe to be hybridized with the analyte)
LC-MS/MSSmall oligonucleotides (e.g., <25 nt)High specificity (for modified RNAs), wide dynamic range, good sensitivity (for modified RNAs), medium to high throughput, good accuracy and reproducibilityCostly instrument
Limited specificity and sensitivity, particularly for unmodified RNAs
  • LC-MS/MS, LC tandem mass spectrometry.