8] Isolation and cell-free translation of human interferon mRNA from fibroblasts and leukocytes

Abstract

Publisher Summary This chapter presents the isolation and cell-free translation of human interferon mRNA from fibroblasts and leukocytes. The isolation of interferon messenger RNA (mRNA) is important for studying the expression and regulation of the interferon genes. The chapter describes a number of different methods for the isolation of active mRNA from a wide variety of sources. In each case, the primary concern has been either the rapid separation of the RNA from nucleases or the rapid inactivation of nucleases. Human fibroblast interferon mRNA has been isolated in a number of laboratories using modifications of phenol extraction procedures. In each case, the mRNA was isolated and, by use of one of a variety of translation systems, shown to code for biologically active human fibroblast interferon. Human leukocyte interferon mRNA is isolated from a lymphoblastoid cell line also by use of a phenol extraction procedure. The isolation of biologically active interferon mRNA from induced human leukocytes has been difficult because of the high levels of nucleases present in these cells. Phenol extraction procedures is proved to be unreliable for the isolation of leukocyte interferon mRNA, the usual result being extensive degradation of the RNA.