Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.
We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.
D000818:Animals; D002478:Cells, Cultured; D003850:Deoxyribonuclease I / Q000302:isolation & purification*; D004331:Drosophila melanogaster; D006493:Heparin; D006801:Humans; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000302:isolation & purification*
D012313:RNA; D006493:Heparin; D003850:Deoxyribonuclease I