I exposed my bacteria to different factors which may affect the expression of a gene. However it has affected the growth of one of my bacteria exposed in a certain stimuli. I can't compare CT values from a medium with a "lot" of bacteria and a medium with "less". I'm thinking on diluting until they will have the same absorbance on the spectrophotometer. But I am not sure if it will be fine. If you have any suggestions, feel free to share. Any help would be greatly appreciated.
If you have another gene known to be unaffected by your treatment, you can use it as internal reference to correct for population differences between your samples
Yes why not. There is no relation between gene expression and population of gene. Coz gene expression is something related to expression or supression of gene system regarding chemical n biochemical actions in organisms.
If you have another gene known to be unaffected by your treatment, you can use it as internal reference to correct for population differences between your samples
Without normalization to a control gene, the Ct values of your target gene are useless.
The correct way to analyze your data would be to normalize the Ct values for your gene of interest to an endogenous control for that individual culture (e.g. 16S rRNA transcripts or another housekeeping gene that is not affected by your treatment), and then to compare the relative expression in each of your samples. Theoretically, your the number of transcripts of your endogenous control should be proportional to the number of organisms, thus eliminating the need to dilute to the same OD, etc.
This is a basic principle of quantitative PCR, and I recommend you read:
http://www.ncbi.nlm.nih.gov/pubmed/11328886?dopt=Citation
which explains the Pfaffl method for qPCR data normalization.
Thank you so much Regier and Sir Thompson! I didn't really realize that until you answered the question! thank you so much for your help. cheers.
It is advisable to use more than one house keeping gene since the conditions you compare might affect even genes that are normally invariant. If you use a series of 3 or more HKG you can calculate a virtual HKG and eventually eliminate outlyer HKGs using Bestkeeper (www.gene-quantification.de/bestkeeper.html)
of course to do your RT you will use the same amount of RNA. Usually is in this way that the experiments are measured. but you can do this and at the same time determine the amount of RNA that is in each bacteria, expressing your data in both ways (per microgram of RNA or the differences per bacteria) which in some cases will give you extremely different data (and sometimes more interesting
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