Obtaining high-quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments. Most RNA isolation products use the powerful chaotropic salt solution guanidinium isothiocyanate for sample lysis and homogenization, followed by organic extraction and alcohol precipitation or solid-phase purification. Organic extraction using acidified phenol and chloroform removes proteins, lipids, and DNA from the RNA sample, which is then recovered by alcohol precipitation. Solid-phase, column-based procedures utilize glass-fiber filters that bind RNA; proteins and DNA are removed by washing them through the filter. RNA is then eluted from the filter with RNase-free water. An alternative to column-based procedures is magnetic beads, which also bind RNA very efficiently under selective conditions.
Figure 1. Total RNA Yield (µg) from Different Tissues (mg) * g total RNA/mg tissue. This is intended as a general guide only and may vary depending on physiological state or organism.
Usually the first step after RNA isolation is to measure how much was recovered — the yield. There are also several methods for assessing RNA integrity and purity, both components of RNA quality, that may affect downstream applications. The following sections discuss aspects of measuring quantity and quality of isolated RNA.