Abstract
Previous studies have reported the use of globin chain-specific complementary DNAs to quantitate the amount of human globin mRNA and human globin genes in normal and abnormal cells. In order to obtain larger amounts and more purified globin mRNAs as templates for these experiments, preparative polyacrylamide gel electrophoresis in formamide has been used to separate alpha- and beta-globin mRNA from polyadenylate containing RNA of human reticulocytes. Fifty to one hundred-fifty micrograms of RNA can be applied to the preparative gel and the recovery of the globin mRNA is about 50%. The isolated alpha- and beta-globin mRNAs were assayed in a wheat germ cell-free system, and the alpha- and beta-globin synthesized quantitated by cellulose acetate electrophoresis. The purified alpha- and beta-globin mRNAs direct globin synthesis which is more than 90% either alpha- or beta-globin, respectively. The cDNAs prepared using each of the isolated mRNAs as template are also shown to be specific for alpha- or beta-mRNA sequences. The gel electrophoresis technique used permits the relatively large scale isolation of alpha- or beta-globin mRNAs from human cells.
Highlights
From the Departments of Medicine and Human Genetics and Development, College of Physicians and Surgeons, New York, New York 10032
We have recently reported the use of human globin U- and P-cDNAs prepared by other methods for measurement of the globin gene content and globin mRNA content of cells from normal and thalassemic subjects [3, 4]
Previous methods using polyacrylamide gel electrophoresis in formamide to separate (I- and /SmRNAs from human cells as well as those from rabbit and mnuse reticulocytes have depended on homogenization of the gel to elute the RNA [6,9, 11]
Summary
From the Departments of Medicine and Human Genetics and Development, College of Physicians and Surgeons, New York, New York 10032. We have recently reported the use of human globin U- and P-cDNAs prepared by other methods for measurement of the globin gene content and globin mRNA content of cells from normal and thalassemic subjects [3, 4] These globin cDNA probes are highly enriched for The methodolo&y described here permits the relatively large scale preparation of a- and /I-mRNAs
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