To profile a bacterial community, we can do pyrosequencing of 16S rRNA genes (the DNA). We can also extract the rRNA and do cDNA sequencing. We assume that the rRNA sequencing measures the active bacteria while the DNA sequencing measure both active and inactive. Are there any association between these two data?
Daniel
In addition to what has already been said. Traditionally DNA was presumed to detect what organisms are present and RNA what organisms are active, although this is clearly not absolute as dead cells can contain DNA, DNA can exist outside cells and different organisms reduce rRNA content at different rates when they become inactive. However, it is a good working hypothesis to assume that detecting an organism using RNA is a good indication of activity if you can also detect its presence using DNA. You cannot infer numbers of cells from either set of data. Critical to any analysis of RNA is a robust and effective RNA extraction method. This will depend on what samples you are working with but if they are environmental samples have a look at my paper in Methods in Enzymology 397 271-292 (2005) which describes the rationale behind extracting nucleic acids.
BrdU has been used to look at activity but it is not taken up by all cells.
rRNA will still exist in inactive bacteria as well, since the ribosomes will stick around for some time. The amount of amplification from rRNA sequencing will probably not correlate well with the number of cells, as ribosome levels may vary from cell to cell. Sequence differences found in either type of sequencing will give you an idea of the species composition of your sample, but the DNA results should provide a better estimate of relative populations.
Dear Daniel, I agree with the two previous posts, the nb of 16S rRNA or rDNA reads won't reflect the number of cells, active or not. Maybe you could incubate your samples (if fresh) with BrDU and then sequence BrDU-labelled DNA (here are some examples of application: http://onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2007.01521.x/abstract or http://www.frontiersin.org/Aquatic_Microbiology/10.3389/fmicb.2013.00118/abstract). And for your information, the patterns of diversity and taxa abundances seem congruent between bacterial 16S rRNA or rDNA when using classical approaches (see http://www.pnas.org/content/early/2012/12/05/1215210110.short and http://www.pnas.org/content/108/31/12776.short)
Daniel
In addition to what has already been said. Traditionally DNA was presumed to detect what organisms are present and RNA what organisms are active, although this is clearly not absolute as dead cells can contain DNA, DNA can exist outside cells and different organisms reduce rRNA content at different rates when they become inactive. However, it is a good working hypothesis to assume that detecting an organism using RNA is a good indication of activity if you can also detect its presence using DNA. You cannot infer numbers of cells from either set of data. Critical to any analysis of RNA is a robust and effective RNA extraction method. This will depend on what samples you are working with but if they are environmental samples have a look at my paper in Methods in Enzymology 397 271-292 (2005) which describes the rationale behind extracting nucleic acids.
BrdU has been used to look at activity but it is not taken up by all cells.
If someone interested in the diversity of active microbial community, a better alternative might be stable isotope probe (SIP) where stable isotope labelled substrate (C-13, N-15 or S-34) are deployed into the environment (eg., soil in fields) and the substrate are taken by only active microbes or functional groups responsible for substrate degradation. After a certain time, extract microbial DNA and fraction heavy DNA from light DNA. The DNAs can be sequenced. The community of heavy DNA represents the active microbes and can be compared with that of light DNA.
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