Detection of HIV-1 Nucleic Acids by Northern Blotting
Northern blotting (1) is one of many tools used in understanding the human immunodeficiency virus type 1 (HIV-1). In the process of Northern blotting, RNA is first separated by size through a denaturing agarose gel and transferred onto a membrane. With this transfer or blotting, and subsequent hybridization with a DNA or RNA probe, quantitation, expression levels of the RNA, size of the RNA, and mapping of the 5′- and 3′-terminal end of the RNA can be determined from primary and cultured cells, blood, and tissue. For HIV research, Northern blotting has been utilized for determining the expression levels and splicing patterns of HIV RNA regulatory genes (2, 3, 4), HIV RNA protease gene (5), cytokine effects on the levels of spliced and genomic HIV RNA (6,7), steady-state transcriptional levels and splicing patterns of HIV RNA as a result of antiviral constructs (8), and receptor studies (9,10). In addition, Northern blotting has be utilized to answer questions on the effect on the HIV RNA level of expression of the envelope protein on calmodulin (11) and carbohydrate binding proteins (12).
KeywordsSodium Lauryl Sulphate Splice Pattern Transfer Buffer Guanidinium Isothiocyanate dsDNA Probe
- 3.Cagnon, L., Cucchiarini, M., Lefebvre, J. C., and Doglio, A. (1995) Protection of a T-cell from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule. J. Acquir. Immune. Defic. Syndr. Hum. Retroviol. 9(4), 349–358.Google Scholar
- 7.Weissman, D., Poli, G., and Fauci, A. S. (1995) IL-10 synergizes with multiple cytokines in enhancing HIV production in cells of monocytic lineage. J. Acquir. Immune. Defic. Syndr. Hum. Retroviol. 9(5), 442–449.Google Scholar
- 12.Schroder, H. C., Ushijima, H., Theis, C., Seve, A. P., Hubert, J., and Muller, W. E. (1995) Expression of nuclear lectin carbohydrate-binding protein 35 in human immunodeficiency virus type 1-infected Molt-3 cells. J. Acquir. Immune. Defic. Syndr. Hum. Retroviol. 9(4), 340–348.Google Scholar