This step is very critical and important for any molecular-based technique since it ensures that the small RNA fragments found in the samples to be analyzed are characterized by a good level of purity and quality. There are different purification methods that can be used, based on the purposes of the experiment: 
* acid guanidinium thiocyanate-phenol-chloroform extraction: it is based on the use of a guanidinium-thiocyanate solution combined with acid phenol that disrupts cell membranes bringing in solution the nucleic acids and inactivating cellular ribonucleases (chaotropic agent). After this step an aliquot of chloroform is added in order to separate the aqueous phase (containing the RNA molecules) from the organic phase (cellular debris and other contaminants).
* spin column chromatography: universally used method to purify nucleic acids that exploits a spin column containing a special resin that, after a first step of cell lysis, allows the binding of the RNA molecules, eluting unbound particles (several proteins and rRNA). The protocol includes two separate chromatographic runs: the first one is required to isolate the whole RNA content from the sample, while the second one is specific for the isolation of small RNA by adding a small RNA enriched matrix to the column and by using a specific buffer to finally elute them. This method can separate small RNA molecules without the need of adding phenol.
Once small RNAs have been isolated, it is important to quantify them and to evaluate the quality of the purification. There are two different methods to do this: 
* analysis of the absorbances and gel electrophoresis: this practical approach exploits the use of a spectrophotometer to evaluate the absorbance of RNA molecules at 260 nm (1 OD = 40 μg/μL) in order to estimate their concentration and to discover possible contaminations (i.g. proteins or carbohydrates); this can be coupled with an electrophoretic run performed in denaturating conditions (8 M urea) to analyze the quality of the purification extracts (low quality extracts will be degraded and displayed as smears in the gel).
* Agilent bioanalyzer: fully automated technique that is based on the use of a special apparatus composed by a chip that allows to perform capillary electrophoresis (CE) using small aliquots of the starting samples and obtaining an electropherogram that is useful to estimate the quality of the extracts thanks to a score (ranging from 1 to 10) attributed by the system.