What is the key technical challenge in isolating high quality RNA from cell or tissue samples?
Ribonucleases are the principal threat to any RNA isolation procedure. In addition, copurification of inhibitory contaminants is a major problem when isolating RNA from certain tissue sources. To minimize the threat, gloves should be worn at all times, and special care must be taken to use RNase-free reagents and labware.
In addition, tissue/cell lysis steps are typically carried out with lysis buffers containing guanidine isothiocyanate, a potent protein denaturant. It is very important to use a sufficient amount of lysis buffer during RNA isolation. We recommend using at least 10x volume of lysis buffer to tissue/cell pellet.
In general, for fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits. It is more challenging to isolate high-quality RNA from tissue samples than from cultured cells, especially those tissues containing high levels of RNase, or difficult-to-homogenize tissues. Examples of such tissues include liver, heart, skin, and conjunctive tissues. Many tissue samples also contain difficult-to-remove contaminants (such as polysaccharides, collagen, fats, lipids or fibrous components) that may interfere with subsequent enzymatic reactions if not removed from the RNA preparation. For purification of high-quality RNA from difficult tissues we recommend QIAGEN’s RNeasy Plus Universal Kit.