Chapter 2 : RNA Stable Isotope Probing

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This chapter focuses on RNA-based stable isotope probing (RNA-SIP) that was developed to take advantage of the features of RNA that make it an excellent biomarker for linking environmental processes—rapid turnover rates independent of cellular replication, coupled to in-depth phylogenetic information within the molecule itself. The primary aim of an RNA extraction protocol for RNA-SIP is to generate over 1µg of quantifiable RNA. Nucleic acids appear in almost all gradient fractions as revealed by high-sensitivity methods for detecting them, such as reverse transcription PCR (RT-PCR) or PCR. The chapter also talks about the RNA-SIP- directed investigation that ultimately led to the isolation of a novel strain responsible for the observed phenol degradation and to confirmation that it could be used to revive sludge that had lost phenol-degrading activity. These findings changed one's understanding of the microbes responsible for phenol degradation in aerated sludge, revealed the pitfalls of both conventional culturing and basic molecular approaches, and highlighted the importance of methods linking function with phylogeny. Manefield used RNA-SIP to compare the communities dominating the assimilation of carbon from phenol in near-identical wastewater treatment bioreactors that were operated in the same manner but differed in wastewater treatment performance. This study revealed that species were responsible for phenol degradation and the poor performance of one reactor was associated with two different populations, while the strong performance of the other was associated with a single dominant lineage.

Citation: Manefield M, Gutierrez-Zamora M, Whiteley A. 2011. RNA Stable Isotope Probing, p 25-36. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch2
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Image of FIGURE 1.

Citations of the original publication of the RNA-SIP method applied to the identification of bacteria dominating the assimilation of carbon from phenol in an industrial wastewater treatment plant located in northeast England. Over seven years the manuscript has been cited a total of 165 times (source, ISI Web of Knowledge).

Citation: Manefield M, Gutierrez-Zamora M, Whiteley A. 2011. RNA Stable Isotope Probing, p 25-36. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch2
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Image of FIGURE 2.

Conceptual relationship between apparent distribution of nucleic acids in equilibrium density gradients using methods with different levels of sensitivity. (A) Apparent distribution of nucleic acids in equilibrium density centrifuge tube if a method with low sensitivity is used (e.g., ethidium bromide staining of nucleic acids within the centrifuge tube). (B) Actual distribution of labeled and unlabeled nucleic acids in equilibrium density gradients as determined by high-sensitivity detection methods (e.g., quantitative PCR or RTPCR). (C) Apparent distribution of nucleic acids in equilibrium density gradient using a detection method with moderate sensitivity (e.g., concentration of nucleic acids by precipitation followed by agarose gel electrophoresis with SYBR gold staining).

Citation: Manefield M, Gutierrez-Zamora M, Whiteley A. 2011. RNA Stable Isotope Probing, p 25-36. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch2
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