What is the key technical challenge in isolating high quality RNA from cell or tissue samples?
Ribonucleases are the #1 threat to any RNA isolation procedure. In addition, co-purification of inhibitory contaminants is a major problem when isolating RNA from certain tissue sources. In order to minimize the threat, gloves should be worn at all times, and special care must be taken to use RNase-free reagents and lab ware. 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 10 X volume of lysis buffer vs tissue/cell pellet (10X l of lysis buffer/mg of tissue or cell pellet). 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 difficu
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- What is the key technical challenge in isolating high quality RNA from cell or tissue samples?
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