Bacterial Transcriptome sample prep following different doses of drug

[firefly2280]

Dear all

If we wanted to look at bacterial transcriptomes and how they differ following exposure to different doses of a drug, how important isi t to keep the bacterial population the same? I mean, if they are exposed to a low concentration, they will grow more but a higher concentration they will grow less. I know you normalise RNA before sequencing but I read you should keep the OD of the culute the same throughout.

It will only be a max of 1 hr, so Im not sure the OD would change too much anyway.

any thoughts?

thanks

[kwaraska]

We always normalize based on total RNA. The transcript population will vary but will be proportional. Keeping the OD of the culture is less important(in my opinion) than starting with equal total RNA

[buthercup_ch]

What is the meaning of "keep the OD of the culture the same throughout"?

You are going to study the differential expression between control and treated samples, right? Then of course bacteria will behave differently depending if they are treated or not.
What is important for transcriptomics is to use high quality RNA samples as starters. Normally 0.5-4 micrograms of total RNA is enough to work with, but I recommend to obtain as much as you can to ensure you will have enough for all QC and further validation of your differential expression results.
For that, the only thing you have to do is standardize the amount of bacteria you have to harvest to proceed with RNA extraction, and in this regard, yes, to keep a "total" OD of your samples is helpful. It means, harvest the same a mount of bacteria treated or untreated.

Hope this will help