1) The PLG tube is not essential, but it does make it easier to remove the aqueous phase without contamination with the interphase.
2) A spike-in control can be added here. For example, add 3 μl of the miRNeasy Serum/Plasma Spike-In Control (the manufacturer’s instructions produce a working solution of 1.6 x 108 copies/μl, so if you add 3 μl of the Spike-In Control and elute your exRNA in a final volume of 30 μl, the theoretical concentration of the Spike-In Control in the final exRNA sample would be 1.6 x 107 copies/μl).
3) The centrifuge must be above 20°C so that excessive precipitation does not occur.
4) After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
5) To avoid damage to their lids, place the spin columns into the centrifuge with at least one empty position between columns. Orient the lids so that they point in a direction opposite to the rotation of the rotor (e.g., if the rotor rotates clockwise, orient the lids counterclockwise). It is important to dry the spin column membrane, since residual ethanol may interfere with downstream reactions. Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution.
6) As little as 10 μl RNase-free water can be used for elution if a higher RNA concentration is required, but the yield will be reduced by approximately 20%. Do not elute with less than 10 μl RNase-free water, as the spin column membrane will not be sufficiently hydrated. The dead volume of the RNeasy MinElute spin column is 2 μl: elution with 14 μl RNase-free water results in a 12 μl eluate.