RT-PCR of 16S RNA

[Jim Sullivan]

Hi all,

Sorry for the mass e-mail, but I just came across a 2004 ref (Rogers et al., 2004, J. Clin Microbiol, 42:5176-5183) wherein they reverse transcribed 16S RNA and then performed traditional phylotyping after producing 16S amplicons from the cDNA. This seems like a reasonable and straightforward method to identifying the fraction of the community that it is transcriptionally active (as compared to 16S typing from DNA wherein non-viable cells and free DNA will also be typed).

I'm a bit surprised that I haven't this method in more widespread use, so I wanted to poll you experts to (i) get a sense of what you think about the method and (ii) to see if there any methodological pitfalls with this approach that aren't obvious to me.

Hope all are well,

Jim


James C. Sullivan, PhD
Associate Director, Clinical Biomarkers
Translational Medical Sciences
Vertex Pharmaceuticals
130 Waverly Street
Cambridge, MA 02139
Tel: 617-961-7536


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[Jonathan Kirk Harris]

Jim,

This approach is more technically challenging, and has it's own issues. RT of the structural RNA is part of the issue. It can be difficult to RT through structures, and it isn't clear how you would validate this approach. The difficulty would be the need to understand how the different types of bacteria fit within the system. I don't think that you gain as much as expected from this approach.

Ribosomal RNA amounts can vary greatly based on growth rate. Not all types of bacteria maintain the same amount of ribosomes based on the growth rate.

Kirk
________________________________________
From: cftellu...@googlegroups.com [cftellu...@googlegroups.com] On Behalf Of Jim Sullivan [james_s...@vrtx.com]
Sent: Friday, August 16, 2013 6:11 AM
To: cftellu...@googlegroups.com; Kevin Kelliher
Subject: RT-PCR of 16S RNA

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