Analytical methods for oligonucleotides or RNAs
| Method | Analytes | Advantages | Disadvantages |
|---|---|---|---|
| qPCR (amplification) | Unmodified oligonucleotides (chemical modifications may affect the assay) | High sensitivity, broad dynamic range (4–8 orders of magnitudes), medium to high throughput | Extensive 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 oligonucleotides | High specificity (sequence dependent) | Depends on the reliability of probe, detection marker, or antibody |
| Wide dynamic range | Possible matrix effects | ||
| Good sensitivity | May not distinguish metabolites from the analyte | ||
| Reasonable accuracy and reproducibility | |||
| LC-UV (or FL) | Modified or unmodified oligonucleotides | High specificity after separation | Extensive sample preparation |
| Good accuracy | Low sensitivity | ||
| Broad dynamic range | Requires good separation | ||
| Low to medium throughput | (Sensitive FL detection depends on the fluorophore-labeled probe to be hybridized with the analyte) | ||
| LC-MS/MS | Small 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 reproducibility | Costly instrument |
| Limited specificity and sensitivity, particularly for unmodified RNAs |
LC-MS/MS, LC tandem mass spectrometry.