Choice of volume for elution is dependent on RNA size and RNA input amount and therefore should be tailored to each sample type and eluate concentration requirement. Re-loading the column multiple times (> 5) during sample loading steps can make elution more challenging; additional elutions will ensure successful recovery.
- A volume of 250–500 µl is recommended for the first elution. It is recommended to use 500 µl nuclease-free water for RNA
sizes > 5 kb. - For RNA sizes < 5 kb at 3 mg RNA input, a second elution with 100–500 μl nuclease-free water is recommended.
- For RNA sizes ≥ 5 kb at 3 mg RNA input, a second elution with 100–500 μl nuclease-free water is necessary.
- For RNA sizes ≥ 9 kb at 3 mg RNA input, increasing the first elution volume to 500 μl and using 250–500 µl for subsequent elutions is recommended. A third elution can maximize recovery.
