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Chapter 40 : Measurement of rRNA Abundance by Hybridization with Oligodeoxynucleotide Probes

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Abstract:

This chapter provides background information and basic methodologies required to carry out the measurement of rRNA abundance by hybridization with oligonucleotide probes. Much of the discussion in the chapter focuses on hybridization to RNA, but many of the methods presented are equally applicable to DNA. The chapter addresses the extraction of RNA from environmental samples, the design of oligonucleotide probes, probe labeling and hybridization, and methods for calculating rRNA abundance from hybridization data. Several methodological studies have evaluated the use of RNA hybridization in microbial ecology and have contributed to many of the protocols and methodological caveats.

Citation: Buckley D, Schmidt T. 2007. Measurement of rRNA Abundance by Hybridization with Oligodeoxynucleotide Probes, p 897-908. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch40

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Figures

Image of FIGURE 1
FIGURE 1

The temperature of dissociation () for an oligonucleotide probe calculated from three replicate experiments. Data are obtained by hybridizing the probe to 1 µg of complementary RNA on a nylon membrane. The probe is then removed from the RNA by gradually increasing the wash temperature. The probe removed is expressed as a percentage of the probe bound to the RNA prior to washing.

Citation: Buckley D, Schmidt T. 2007. Measurement of rRNA Abundance by Hybridization with Oligodeoxynucleotide Probes, p 897-908. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch40
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Image of FIGURE 2
FIGURE 2

Effect of humic acid contamination on rRNA hybridization. A constant amount of target RNA (60 ng) was added to increasing amounts of a soil RNA extract that was heavily contaminated by humic acids. Hybridization with a radiolabeled oligonucleotide probe that bound only to the target RNA was performed on the target RNA spiked with soil RNA extract, soil RNA extract alone, and target RNA alone. Hybridization of probe to target RNA decreased with increasing amounts of the contaminated soil RNA extract.

Citation: Buckley D, Schmidt T. 2007. Measurement of rRNA Abundance by Hybridization with Oligodeoxynucleotide Probes, p 897-908. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch40
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References

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Tables

Generic image for table
TABLE 1

Final stringent wash temperatures for a variety of oligonucleotide probes

Stringent wash temperatures for a selection of commonly used rRNA targeted oligonucleotide probes in buffer lacking formamide as described in section 40.4.2.

Citation: Buckley D, Schmidt T. 2007. Measurement of rRNA Abundance by Hybridization with Oligodeoxynucleotide Probes, p 897-908. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch40
Generic image for table
TABLE 2

Example calculation of rRNA relative abundance for a single RNA sample

Data are from one hybridization experiment with a universal probe and a second hybridization experiment with a group-specific probe. The hybridization experiments were carried out on duplicate membranes that contained RNA from a positive control organism, a negative control organism, and a soil sample. Five different volumes of each RNA sample were blotted on each membrane. With a 96-well blotting apparatus it is possible to create a single membrane that contains 19 different samples and controls.

Citation: Buckley D, Schmidt T. 2007. Measurement of rRNA Abundance by Hybridization with Oligodeoxynucleotide Probes, p 897-908. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch40

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