I want to compare transcription of a gene both inserted in the genome and a plasmid. Should I extract the RNA in the exponential phase or stationary phase?
Hello, mRNA usually expressed in the stationary phase, which was detectable due to the addition of autoinducer molecules in the exponential phase. The better to isolate in stationary phase.
OD at least 0.8 at 600nm better above 1.0 depends from inoculum.
I have problem with your answer.
Do you consider that bacteria use different genes in different phases of growth?
Do you check your gene about this?
No different genes. In stationary phase RNA expression is higher and extraction in this phase is better to have higher yield of RNA.
I do not agree with the stationary phase isolation. But of course, it depends what is your biological question to be answered. If you want ot compare the GFP expression on both a plasmid and the chromosome, then their expression depends on the promoter that drives them. Generally speaking gene expression is more intense in the exponential phase than in the stationary! I would recommend to isolate RNA at OD 0.5-0.6. Or you can do it at several time points to compare. Do not forget to stop growing by adding 4 ml stop solution (5 % v/v phenol in abs ethanol) to 30 ml culture, to stop growing and inactive RNAses.
Hello, gene expression is stabile in stationary phase you will have good repeatability in stationary phase, also in this phase you will have maximum number of cells so maximum quantity of plasmids. How to study gene expression in stationary phase see below link. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096701
I agree with @Ferenc Marincs, it would depend on what genes/promoters that you want to study and the purpose of your experiments. It's probably a good idea that you look at gene expression profiles at different cell densities (OD readings) including stationary cells, and under different growth conditions. There are a large body of published data in the field, for example, see The ISME Journal (2015) 9, 1130–1140; Nucleic Acids Research, 1999, Vol. 27, No. 19 3821–3835; THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 32, Issue of August 8, pp. 29837–29855, 2003. There are others. So, you may want to check the published studies to see if people already published on the gene/promoter that you would like to test, which would save you time and efforts. I think your gene/promoter of interest should be in these whole genome data files.
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