If I have a gene carried on a plasmid and the plasmid is transformed into two strains of different bacterial species (Salmonella and e. coli). If I want to do RT-qPCR to quantify the expression of that gene in the two strains. what is the best way to normalise the assay? or what is the best reference gene could be used in this situation? Thanks in advance.
Osama Kamal the best way is to test the common reference gene in function of ypur study. Because you need to find a gene that it is not affected from your experiment, means that its quantification remain stable during all your experiment.
As you are studying the gene transcription from plasmid, I think you can use the antibiotic resistance gene, present in the plasmid (as selection marker), I believe you are using same plasmid in different bacterial strains.
In general house keeping (GAPDH, 16s rRNA) genes are used for normalization.
Giorgia Pertile Thanks a lot for your reply. The trick is that for E. coli on its own there are dozens of reference genes and the same for salmonella on its own. But to compare both, I think I need to find a gene which is expressed at the same level in both species which is difficult to find and tricky!!
Thanks Mantu Kumar for your reply. Yes I am using the same plasmid in both strains and I was thinking of using one housekeeping gene in addition to the resistance marker. But I am still not sure if the copy number of the plasmid would differ in two different bacterial species?!
@Osama Kamal You need to read bibliography and try in your experiment which is the best
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