The selective enrichment of rough endoplasmic reticulum (RER) fractions obtained from secretory tissues provides for a highly-efficient method for purifying mRNA molecules destined to be used as templates in the synthesis of secretory proteins. We have shown that the use of a non-ionic, low- viscosity linear gradient fractionates polysomes/free ribosomes and RER in distinct regions of the gradient, in accordance with their predicted densities. Two distinct systems were tested for the applicability of this method. In the case of mouse liver, rat serum albumin and rat actin, radioactively-labeled cDNA probes were shown to hybridize to distinct fractions of the gradient on Northern blots. mRNA from the four fractions with the highest albumin signal was isolated from oligo dT columns and used to synthesize cDNA, which was shown to have high-labeling activity with the albumin, but not the actin probe. In addition, RER from three human-derived, liver cancer cell lines was purified on the same type of gradient, and the separation of polysomes/free ribosomes from RER was again demonstrated with radioactively-labeled human cDNA probes. Our next step is to use a liver cell line or cancer tissue RER mRNA to construct subtraction libraries which will be screened for secretory tumor markers. We hope to expand this technique to other types of tissue in the future.
