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Endogenous control in bacterial RT-qPCR


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#1 front_hands

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Posted 01 September 2009 - 10:48 AM

Hi,

I am looking at gene expression in a particular bacterium under different growth substrates. I am trying to find an endogenous control to normalise my target gene expression levels to. I have tried 16S rRNA but it appears to be too variable depending on the different growth conditions of the bacteria (stastically different). I wanted to try a number of other genes, e.g. rpoB, gyrB, recA, etc, however I have been advised that 16S rRNA should be fine to use as long as we accept the limiations of it's variability.
There is not a genome sequence of my bacterium so I do not have the gene sequences fro easily available specific primers.
Any ideas on what I could do?

I have also had the suggestion that as I am using nanodrop to quantify my RNA after extraction, this could skew my results if it is not reliable enough (I use the same amount of RNA into the RT reaction and then the same amount into the PCR reaction).
Any ideas on where I should go next, or experience with nanodrop vs other measures?? I do not have access to a bioanalyzer but am considering pico green?

thanks!!!!

#2 fishdoc

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Posted 01 September 2009 - 11:08 AM

Have you tried multiple sets of 16s primers? I've found that different 16s primer pairs work better than others, give better efficiencies, and better reproducibility.

How much variability is there in your current set? Sometimes "statistically different" does not equate to "biologically significant".

#3 front_hands

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Posted 01 September 2009 - 01:21 PM

Thanks for the suggestions. I have only tried one set of species specific primers so I can try some others.

How would I tell the difference between statistically significant and biologically?
(They grow better in the no treatment control - but would using the same amount of RNA/cDNA cancel that out?)
In one experiment, the Ct's for 16S rRNA were 24.8, 20.3, 18.8, 17.5, 16.4 and 16.7 for growth at time 0, 24, 48, 72, 92, and no treatment control.
In another experiment no treatment control Ct was 16, while treatments were 17.4, 16.6, and 20.4

Thanks heaps :)

#4 fishdoc

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Posted 01 September 2009 - 01:58 PM

Thanks for the suggestions. I have only tried one set of species specific primers so I can try some others.

How would I tell the difference between statistically significant and biologically?
(They grow better in the no treatment control - but would using the same amount of RNA/cDNA cancel that out?)
In one experiment, the Ct's for 16S rRNA were 24.8, 20.3, 18.8, 17.5, 16.4 and 16.7 for growth at time 0, 24, 48, 72, 92, and no treatment control.
In another experiment no treatment control Ct was 16, while treatments were 17.4, 16.6, and 20.4

Thanks heaps :)




If those Cts are for experiments in which you're adding the same amount of RNA to the cDNA synthesis reaction in each reaction, then it's definitely biologically significant. Are you treating your RNA samples with DNase prior to using it?

#5 front_hands

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Posted 01 September 2009 - 02:07 PM

Yes I am treating the samples with DNase prior to the RT reaction and confirming this with a standard PCR. I also use no RT controls in the qPCR.

Edited by front_hands, 01 September 2009 - 02:09 PM.





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