Abstract
There are several advantages to using an in vitro system for studying mRNA turnover. The polysome is the most important component of the cell-free mRNA decay system we have described. It contains both the mRNA to be degraded and the enzymes and cofactors that degrade the mRNA. The major criterion used to assess the reliability and fidelity of the system is the assay for relative decay rates of different mRNAs. Solutions are made with ultrapure reagents using double-distilled water. Buffers containing tris(hydroxymethyl) aminomethane (Tris) are adjusted to the indicated pH at room temperature with HC1. The centrifuge tubes are inverted for several minutes (in a cold room), to allow buffer to drain away from the polysomal pellet. The polysomal pellet is rinsed twice with 0.5-ml portions of buffer A, which are then discarded. An additional 0.5 ml of buffer A is added, and the pellet is resuspended by repeated up and down pipetting with a micropipet.