1 Adapted from miRNeasy serum/plasma kit – added 5x volume of Qiazol.
2 PLG makes separation of the aqueous phase from the interface easier, and thus is particularly useful for large numbers of samples or less experienced personnel, but it is expensive.
3 If there is poor phase separation, the original biofluid can be diluted. To “rescue” the exRNA preparation, additional buffer, Qiazol and chloroform can be added (maintaining the ratio 1:5:1).
4 The manufacturer’s protocol is for 8000 x g, but some labs have found that 1000 x g for the binding step gives better results.
5 The centrifuge must be above 20°C so that excessive precipitation does not occur.
6 80% ethanol should be prepared with ethanol (96–100%) and RNase-free water.
7 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.
8 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.
9 This volume was selected to match that of other kits to enable fair comparisons. 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.
10 Centrifuging at a low speed first helps the solvent wet the surface of the membrane prior to the full-speed centrifuging step. This results in a better yield/RNA recovery from the membrane.