A few factors should be taken into consideration for ~1 mg RNA input before choosing multiple 500 µg columns vs. a single 3 mg column:
- Input Sample Volume. If the starting RNA input volume > 800 µl, it is advisable to use multiple 500 µg columns due to their compatibility with a vacuum manifold. The 3 mg columns are not compatible with a vacuum manifold and require centrifugation for sample loading.
- Required Eluate Concentration. 500 µg columns are compatible with elution volumes 50–100 µl (for ~1 mg input, yielding ~5–10 µg/µl eluate concentration). 3 mg columns are compatible with elution volumes 250–500 µl (for ~1 mg input, yielding in ~2–4 µg/µl eluate concentration).
- Inaccurate quantification of starting concentration. If the starting sample concentration or input amount is not accurately known, such as RNA synthesized using high yield/scaled-up in vitro transcription (IVT) reactions, the 3 mg columns provide the flexibility to capture input amounts > 500 µg in one column, without the risk of inadvertent RNA loss.
