What would be the acceptable CV% and SD for copy number results between stool samples in qPCR (same group)? I am evaluating different bacteria (E.coli, Lactobacillus, etc.) using the absolute quantification method. My CVs after calculating the number of copies was very high (some over 25%). I read that for gene expression this would be acceptable, for bacterial quantification would it also be? If you can give me a reference, I appreciate it. I know that there also doesn't seem to be a consensus on the expression of the final result, I saw articles expressing the result in number of copies, log10, number of copies / ng of feces, among others
I am not giving you the answer but just curious..
If you are getting results which you assume is not acceptable, what you are going to do then, may be based on answers from here? Would you manipulate your results to fit somewhere which could be acceptable?
Can I ask when you say between stool samples in the same group is that group the same person/animal? Are they physically the same stool divided etc?
I think even in the same group if they are different stools or samples over a course of a day/hour etc then a high CV would be expected. Due to the exponential nature of PCR a small difference in target material at the start can lead to a large endpoint difference which I would imagine you would get with something as chaotic as stool.
What I dont know the answer to, which may help, is if there is a standardising element you could use? Such as the was GADPH, Beta Actin etc are used as standards in other work.
Could streaking a plate with a small volume of stool and calibrating to CFU's be an option (just brainstorming in this)
Hope it helps
Abhijeet Singh if i wanted to manipulate my results i'm not even asking anything here. In my laboratory we work with a 15% CV, but I have read in some studies that, in PCR the results can vary a lot, in especial with environmental samples. I saw studies accepting 20%, but I was in doubt about it. Anyway, I will repeat some tests. But I just wanted the opinion of other professionals, that's why I asked here
Stephen Reynolds They are animals from the same group that have had the same treatment over a period. 5 different animals receiving the same treatment. As I am doing PCR with absolute quantification I cannot use a control gene like beta actin, but I can see if I test this from the CFU that you said or something similar, thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA