The NZY miRNA Isolation & RNA Clean-up Kit is engineered for the purification of total RNA, including miRNA, from cellular material and may be used for general RNA reaction clean-up, with the optional isolation of protein and DNA. The sample is stabilized after the initial sample lysis in Buffer NMiL, devoid of RNase activity. Mechanical disruption may be required for hard-to-lyse samples. Lysis Buffer NMiL features denaturing salts and β-mercaptoethanol, aiding in sample lysis and stabilization. Unlysed sample remnants are filtered out, reducing lysate viscosity. Concurrently, high molecular weight DNA is sheared, preparing it for a more efficient DNase digest. The addition of ethanol creates optimal binding conditions for RNA and DNA fragments over approximately 200 nt to the NZYSpin RNA Column (blue rings). Meanwhile, smaller RNA and proteins are collected in the flowthrough. This separation ensures superior RNA purity, as DNA and proteins are separately and efficiently removed. If total nucleic acid purification, including DNA, is sought, on-column DNAse digestion is omitted. Otherwise, a desalting step preps the NZYSpin RNA Column for the on-column DNA digest. Meanwhile, Buffer NMiPP is added to the flowthrough to precipitate proteins, which are then removed via filtration through an NZYSpin Protein Removal Column (white ring), leaving only small RNA. This is combined with Buffer NMiB, which adjusts binding conditions for small RNA to the NZYSpin RNA Column (blue rings). After DNA digestion, this mixture is loaded into the NZYSpin RNA Column (blue rings). If separate fractions of small and large RNA are desired, the mixture can also be bound to a second, novel, NZYSpin RNA Column (blue rings). After stringent washing steps, pure RNA is eluted in 30 – 100 μL of RNase-free water and is ready for both standard and advanced downstream applications.