PURIFICATION OF FUNCTIONAL RNA FROM HUMAN-GRANULOCYTES

Abstract

The presence, in granulocytes, of high levels of nuclease activity makes it difficult to isolate intact RNA from these cells. A method was developed that allows purification of functional RNA from normal granulocytes as determined by capability for reverse transcription and in vitro translation. A considerable amount of ribonuclease activity remains in granulocyte lysates, even after the addition of heparin or vanadyl ribonucleoside complexes. RNA isolated from such lysates demonstrates only minimal binding to oligothymidylic-cellulose and does not serve as a template for reverse transcription or in vitro translation. The extraction of frozen granulocytes into phenol in the presence of both heparin and vanadyl ribonucleoside allows the purification of relatively large quantities of RNA, which serves as an excellent template for reverse transcription and in vitro translation. Purification of granulocyte RNA by this method wil facilitate study of granulocyte gene expression.