We have used 16 S rRNA seq to look at differences in gut microbial composition between patients and controls. Now we want to use qPCR to verify the findings, however, I have only used qPCR before for gene expression, where you use a reference gene to normalise to in order to do relative quant. What do you use when you want to quantify microbial levels?
Stefanie,
Presumably, you have 16S primers "specific" for different groups like Bacteroidetes, Firmicutes, Actinobacteria etc. and you are interested in verifying differences in their levels between different samples. One approach is to use a "universal" 16S primer pair to generate a reference. This should give you a measure of the "total" 16S DNA in each sample (which may or may not be closely correlated with the weight of the original stool sample--you could look at that). Then you are essentially comparing the proportion of each specific group within the total of all 16S sequences in that sample. This approach is similar to the sequencing approach, which also measures the proportion of each group in the total sample without giving any information on the absolute number of each type. A general problem with this approach is the specificity of the primers. Ideally, you would include controls for every type of bacteria that you are measuring and assess the degree to which your primers are specific for that type. This may be a particular concern if you are trying to measure a type that has a low relative content in the total mix and the primers you are using will also weakly amplify a type of bacteria that is common in the total mix. You will not be able to detect a specific signal.
Hi Stefanie,
Definitely you can use qPCR to quantify the microbes...There will be few concerns...first the gene should be of 'reference gene type' so that it should not very in expression levels. (alternatively you may use directly the genomic region for qPCR based microbe level estimation)....however for each microbe the region should have some variability (or the amplicons should be microbe specific) to identify one from other...or else the signal will be mixed up...considering these issues the SNP (from RNA sequencing data) based probes may be a better strategy to quantify the microbes in a specific manner
bye
Dear Stefanie
to solve your problem you should do an absolute quantification of your bacterial target. To do this you should prepare a calibration curve using different amount of genomic DNA extracted from a known amount of bacterial cells (a reference species you can easily cultivate in lab). This should allow you to calculate a cell concentration (n. of cells / g of stool sample) starting from the Ct value obtained from the qPCR assay. Of course the quantification will be affected by the approximation that you builded a calibration curve using a defined species characterized by a defined n. of ribosomal operon in the chromosome, whereas you probably do not know the n. of ribosomal operon per genome in the target species you are going to monitor by qPCR.
If you need further help do not hesitate to contact me
regards
Diego
Thanks so much Diego! So for example you can use an E.coli strain as the reference species? Also, regarding the gram of stool, our stool samples are placed in a preserve fluid right after collection, so I am not sure how to incorporate that in the calculation. We use 1.4 ml of homogenised stool (in this preserve fluid per extraction), should I work on that?
yes, you can use an E coli strain as reference and you can refer to a defined volume of homogenized stool sample ..
I hope all will work fine
by
Diego
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