First, I am thinking about extracting RNA from the same number of bacteria from 2 differents conditions.
Secondly, there would be a quantification and normalization step of RNA ammounts? In this part I am having troubles, what if they dont have similar RNA levels?, there will always have to be a step in wich same ammounts of RNA (normalized) have to be taken in order to proceed to cDNA synthesis?
the later steps are more obvious since many books and protocol manual have described them.
please help about the second part!
thank you
Hi Greg,
This question is not very clear to em but I suspect you need some RNA normalization.
usually, to normalize a specific RNA from one gel lane to the next (for instance), the RNA band corresponding to a mRNA encoding a housekeeping protein is used .. actin for instance. It is assumed that such a mRNA is constant whatever the conditions. Alternatively, you can use a normalization based on ribosomal RNAs.
Best regards,
Philippe
For cDNA synthesis, we use 1 ug of total RNA. That will bring all your samples (in spite of varying concentrations) to a standardized value.
As far as quantifying and normalizing your genes of interest, why not try GAPDH? First step in glycolysis, don't need mitochondria to do it, so this will work well in prokaryotes. GAPDH qPCR or rtPCR primers are widely used, a literature search should get you some very specific ones,
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