RNA/DNA purification and extraction are two important procedures in molecular biology that aims to isolate RNA/DNA from its cellular source and protect it from degradation by cellular enzymes. The procedure is usually done in laboratories for the purpose of subsequent viral and forensic analysis to ensure accurate assays, especially for biological samples like skin, hair follicles, semen, and other tissues. By purifying RNA/DNA, investigators are able to determine what the person looks like, identify the color of the hair, eyes, and skin, and distinguish many other features of an individual.
There are several ways to purify RNA/DNA samples and they vary depending on the nature of the samples and the downstream application.
Also known as cell lysis, this method of purifying DNA involves breaking the cell open through blending or grinding in order to expose the DNA. This can also be done through sonicating the sample, a method of applying sound energy, such as ultrasound using an ultrasonic bath or probe to disturb the particles in the sample. Another type of cell lysis includes removal of membrane lipids by adding detergents or surfactants, or removal of proteins by adding protease – an enzyme that conducts proteolysis. One of the most commonly performed cell lysis for RNA purification is by adding RNase, a type of nuclease that breaks down RNA into smaller pieces.
This is one of the most commonly used techniques, which involves concentrating and de-salting the nucleic acid to extract the RNA/DNA from the cellular base. To do this, laboratories add salt and ethanol to the aqueous solution, which force the nucleic acid to separate from the solution. Other process of alcohol precipitation includes ethanol precipitation of small fragments and Isopropanol precipitation for PCR purification.
Oftentimes abbreviated as PC or PCIA, Phenol–chloroform extraction is a form of liquid-liquid extraction. In this process, chemists form a biphasic mixture or emulsions (i.e. vinaigrettes, milk, and cutting fluids) by mixing an equal volume of phenol-chloroform and an aqueous sample. The RNA is recovered as the ethanol precipitates from the aqueous phase while the DNA can be located as the choloroform separates the solvent in two forms, exposing two colors: a clear, upper aqueous phase (containing the nucleic acids) and a bright pink lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform). This method is only used for DNA purification and is not applicable for short RNA species, such as siRNA, miRNA, gRNA, and tRNA – RNAs that only contain less than 200 nucleotides.
This technique is a type of solid based extraction method for quick purification of nucleic acid. During the In this process, the RNA/DNA sample is added to the column or solution containing denaturing agents to allow the nucleic acid to bind with the solid phase silica depending on the pH level. The column is then washed using a buffer or water.