HK gene overabundance - (Jun/15/2006 )
I am attempting to perform relative quantitation of bacterial gene products and am using the 16S rRNA as my HK gene. The genes of interest are single copy and not abundantly expressed. Therefore, when I run my samples I detect my gene/s of interest around 16-21 cycles whereas the 16S RNA is too concentrated and can not be accurately detected wihtout diluting out the sample. If I dilute out the sample, I then have difficulties detecting my gene/s of interest. Has anyone else experienced this problem and if so what is the most appropriate way to deal with the problem? Thanks so much!
Dear Kalee,
I am in a similar situation! How much cDNA do you use?
I am using approx. 100ng per assay, and I find that my target gene only amplifies very late, around 34 cycles, while my HKG (28S rRNA) amplifies around 13 cycles.
I can try to add more cDNA, but I am not sure that increasing it to say 150 or 200 ng will have very much affect. There is no way I can add 1000 ng per assay!
Have you worked out a compromise?
I am in a similar situation! How much cDNA do you use?
I am using approx. 100ng per assay, and I find that my target gene only amplifies very late, around 34 cycles, while my HKG (28S rRNA) amplifies around 13 cycles.
I can try to add more cDNA, but I am not sure that increasing it to say 150 or 200 ng will have very much affect. There is no way I can add 1000 ng per assay!
Have you worked out a compromise?
I have been doing a one-step reaction with anywhere between 40ng - 1000ng of RNA. I am now going to move to a two-step reaction and will likely use less than 70ng of cDNA. I found a post from a while ago where the person did use different dilutions (amounts) of cDNA for the target and HK/reference gene. The only thing I worry about with this is the accuracy of my dilutions. I have 10 samples I am comparing and if my dilutions are off then it could dramatically effect my results.
I don't know the best way to "fix" the problem (so if anyone has any thoughts they are welcome). I am going to make multiple dilutions of the same sample and then assess the variance between these (to see if dilution would even work). Additionally, I am pursuing a new HK gene (rpoB - the beta polymerase). That is a single copy gene so it may have a Ct value closer to my target. If you find a way around this problem please let me know and I will keep you informed. Thanks!
Hi Kalee,
thanks for that. So its possible to use different amounts of cDNA for the HKG vs target in singleplex reactions ? Thats interesting. I might try and find out more about that.
cheers and good luck
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I have been doing a one-step reaction with anywhere between 40ng - 1000ng of RNA. I am now going to move to a two-step reaction and will likely use less than 70ng of cDNA. I found a post from a while ago where the person did use different dilutions (amounts) of cDNA for the target and HK/reference gene. The only thing I worry about with this is the accuracy of my dilutions. I have 10 samples I am comparing and if my dilutions are off then it could dramatically effect my results.
I don't know the best way to "fix" the problem (so if anyone has any thoughts they are welcome). I am going to make multiple dilutions of the same sample and then assess the variance between these (to see if dilution would even work). Additionally, I am pursuing a new HK gene (rpoB - the beta polymerase). That is a single copy gene so it may have a Ct value closer to my target. If you find a way around this problem please let me know and I will keep you informed. Thanks!
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