Integrity Assessment of Nucleic Acids

The assessment of nucleic acid degradation levels is essential to guarantee the accuracy and reproducibility of the experimental results. This step becomes more important when working with fixed and paraffin-embedded (FPE) tissues, where the variable and unpredictable effects of the fixation processing on macromolecule preservation are dependent not only on the intrinsic properties of the fixative but also on the tissue-related preanalytical variables. In this chapter, several methods for the assessment of nucleic acid integrity are described, the focus being especially on their accuracy in FPE and suitability according to the availability and nature of the starting material. In particular, some PCR-based assays have also been reported as useful methods for integrity estimation when DNA/RNA starting material is very poor. Moreover, special attention has been given to RNA quality assessment, which commonly relies on methods based on the detection of ribosomal RNA as an indirect measure of target mRNA integrity. In this context, the limitations of these approaches in the quality assessment of RNA extracted from FPE have been pointed out, suggesting that PCR-based strategies, based on the detection of specific mRNA targets, still remain the best methods for the analysis of RNA isolated from archival material.