Journal of Methods Microbiological Journal of Microbiological Methods 33 (1998) 115–118 Isolation of RNA from the filamentous fungus Mucor circinelloides b c a, * Muhammad Mukhtar , Zahida Parveen , David A. Logan a Department of Biological Sciences, Microbial Physiology Laboratory, Clark Atlanta University, Atlanta, GA 30314, USA b Division of Infectious Diseases, Thomas Jefferson University, Philadelphia, PA 19107, USA c University of Agriculture, Faisalabad, Pakistan Received 8 January 1997; received in revised form 27 April 1998; accepted 27 April 1998 Abstract Isolation and purification of biologically active RNA from filamentous fungi is difficult because of the complex cell wall and the presence of intracellular ribonucleases in these microorganisms. We have developed a procedure for the rapid isolation of intact RNA from the various morphological forms of the filamentous fungus Mucor circinelloides. Fractionation of fungus RNA by agarose gel electrophoresis revealed distinct bands of ribosomal RNA. The integrity of the RNA was further substantiated by Northern blot analysis by probing it with the 1.9-kb and 2.5-kb EcoR1 fragments of the putative transcription factor gene (MTF1) and the 25S ribosomal RNA gene, respectively. The RNA isolated by this procedure appears to be relatively pure since it has a ratio of absorbance at 260 / 280 of 1.7–1.8. This procedure should be useful for isolating RNA from other filamentous fungi and therefore, will serve as an important tool for the molecular analysis of these organisms.  1998 Elsevier Science B.V. Keywords: Mucor circinelloides; Ribosomal RNA; RNA isolation; Transcription factor Isolation of intact RNA is critical for a number of envelope composition and thus, making isolation of molecular genetic analyses such as Northern blotting, functional RNA more tedious. Therefore, in our cDNA production and transcriptional output quanti- study of gene expression during morphogenetic tation. Most of the RNA extraction procedures changes of M. circinelloides, the isolation of high- depend on simultaneous disruption of cells and quality RNA tended to be a major problem. We inactivation of endogenous ribonucleases report here a procedure for the rapid isolation of (Chomczynski and Sacchi, 1987). In the polymor- RNA from different morphological forms of M. phic fungus Mucor circinelloides f . lusitanicus isola- circinelloides. The method has been developed by tion of RNA has been difficult due to the tough cell modifying the previously described protocols for envelope and high levels of intracellular ribonu- RNA isolation (Orlowski and Sypherd, 1978; Stol- cleases. Moreover, there are different morphological tenburg et al., 1995). The value of this method is that forms of M. circinelloides i.e. spores, budding yeast it should be suitable for isolating RNA from other and hyphal forms (Fig. 1) each having different cell filamentous fungi as well. Mucor circinelloides f . lusitanicus ATCC 1216B * was used in the experiments reported here. The Corresponding author. Tel.: 11 404 880 6830; fax: 11 404 880 6756; e-mail: dlogan@cau.edu growth medium (YPG) contained (wt/vol) 0.3% 0167-7012 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0167-7012(98)00047-5