Nucleic Acid Sample Preparation
Traditional nucleic acid sample preparation methods involve phenol-chloroform extraction, differential precipitation, ethanol precipitation, or in-gel separation. Modern sample preparation methods are based on solid-phase separation and offer easier protocols, demand less hands-on time, are automatable, and minimize the use of toxic chemicals.
The choice of method depends on the type of nucleic acid, the intended downstream application, and the requirements for yield, quality, purity, and scale.Tissues are usually disrupted mechanically or chemically, and the method depends on the tissue type. Cell lysis is a critical step, and it is very important to inactivate nucleases that may degrade genomic DNA or RNA.
Many workflows demand changes in buffer conditions or concentration between steps. This is most often achieved using traditional methods, such as ethanol precipitation or using spin columns, prepacked with gel filtration chromatography media for separation based on size.
Bacterial cells are harvested by centrifugation, lysed, and plasmid DNA is preferentially bound to membranes or solid-phase media like anion exchange or silica resins. Washing steps remove contaminants such as carbohydrates and proteins before purified plasmid DNA is eluted. The yield and purity of isolated plasmid DNA is influenced by cell culture density, duration of growth, culture medium, type of plasmid (high or low copy number), size of the insert, and host strain used.
Most reagent kits are based on silica or anion exchange solid-phase media. Each is amenable to automation, driven by centrifugation or vacuum. Contaminating RNA and protein can be removed by enzymes. Another option, using FTA technology, immobilizes and stabilizes the genomic DNA on a chemically-treated filter card, denatures proteins, and provides long-term storage of the purified DNA at room temperature.
Using a technique called Whole Genome Amplification, large amounts of genomic DNA can be prepared from as little as 10 ng of purified DNA.
The trend is towards purifying total RNA rather than mRNA due to improvements in purification methods and the sensitivity of downstream methods. Most of the current methods are solution- or silica-based and generally involve a chaotrope, or other chemical to inactivate RNases. For example, guanidinium salts inhibit RNases and promote binding of both DNA and RNA to silica. DNases are used to remove contaminating DNA.
Poly-adenylated mRNA can be specifically isolated from eukaryotes by affinity chromatography using a solid phase conjugated with oligo-dT.
It is vital to minimize the level of RNases in the laboratory and reagents. Also, samples must be flash-frozen to ensure that the RNases in the tissue do not degrade RNA, especially in expression studies.