Standard curve needed? - (Feb/20/2008 )

Hi all!

I'm working on soil samples which are all from the same field. I want to investigate if fields with different management practice contain different amounts of bacterial groups. I'm using Primers targeting All Bacteria and 6 different bacterial groups. I want to compare the Ct's of the bacterial groups with the Ct of All Bacteria to determine the percentage of the bacterial groups (100/2^DeltaCt). Because the DNA I'm using is from the same soil I'm wondering if I have to make a standard curve?
If yes, should I make a standard curve for every primer-set with PCR-amplified DNA segments (which were generated with the same primers)? And if I have a standard curve what will it tell me for my analysis, because I'm not interested in copy numbers but in relative amounts.
Looking forward to your answers

Fraggle

Hello,

I think it is a good idea to do a standard curve as a start and see if you can use the deltaCt approach. If the efficency of the primers are different and the slopes of your curves are different than delta Ct will not be reliable. (If the 2 curves are not parallel to one another, you can get a higher or lower dCt depending on at which point you compare the two. If they are parallel (in lots of cases when the primer-probe set is good and works well on your samples), then the difference is always the same no matter at which point you look at it.
Therefore I warmly recommend you to check whether you can use dCt or not by using standard curve. Standard curve does not have to be accurate in terms of copy numbers, you can use a series of dilutions without knowing the exact copy number and get only relative amounts in the end and compare these with one another.