General remarks on RNA handling, storage and stabilization
Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA.
In order to ensure accurate gene expression analyses, it is important that the RNA analyzed truly represents the in vivo gene expression of the sample. This is complicated by the fact that changes can occur during handling of the sample and isolation of the RNA.
Once a biological sample is harvested, its RNA becomes extremely unstable. There are two major types of artifacts that can occur. Downregulation of genes and enzymatic degradation of RNA result in an artificial reduction of both nonspecific and specific mRNA species. At the same time, certain genes can be induced during handling and processing of the sample. The combination of these two effects can result in a transcription profile that differs from the true in vivo gene expression pattern. Immediate stabilization of the RNA expression pattern is a prerequisite for accurate gene expression analysis.