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Applied and Environmental Microbiology, October 2000, p. 4547-4554, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Presence of Humic Substances and DNA in RNA Extracts Affects Hybridization Results

Elizabeth Wheeler Alm,1,2,* Dandan Zheng,1 and Lutgarde Raskin1

Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,1 and Department of Biology, Central Michigan University, Mount Pleasant, Michigan 488592

Received 7 April 2000/Accepted 19 July 2000

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.

* Corresponding author. Mailing address: Department of Biology, Central Michigan University, 157 Brooks Hall, Mount Pleasant, MI 48859. Phone: (517) 774-2503. Fax: (517) 774-3462. E-mail: Elizabeth.W.Alm{at}cmich.edu.

Applied and Environmental Microbiology, October 2000, p. 4547-4554, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

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  • Gulledge, J., Ahmad, A., Steudler, P. A., Pomerantz, W. J., Cavanaugh, C. M. (2001). Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria. Appl. Environ. Microbiol. 67: 4726-4733 [Abstract] [Full Text]  


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