Protocol Online logo
Top : Forum Archives: : Real-Time PCR

transgene ct comparison - help (Feb/03/2007 )

Hi,

I am trying to looking at the expression of a transfected gene uder the control of different promoters. The guy that is showing me how to use Real Time pcr gave me the attached template to compare my transgene expression. however the method he uses has one sample (sample A ) as the control to which every other sample is compared to ie fold increase from sample A). I dont think this can work for me because I am not looking at a different condition, and my parental cells obviousy will not express any transgene because they have not been transfected. And I am doing this with different cell lines so I need a method using ct that I compare using GAPDH within each sample.

I have ran my pcrs with GAPDH for all samples. Can somebody please tell me how I can show this data with regards to expression of GAPDH?

I have also played with trying to get exact copy number using standrd curves. Although my standard curves are a bit crazy, someone else recomended serial diluting my standards with mouse genomic DNA or salmon sperm so that I have a constant level of DNA? Does anyone here do this and could you tell me what you use and how you use it?!

Thank you in advance...

jen

-jenifur-

Hi jen,

you can calculate delta ct values for every preparation using GAP-DH. Then you can compare the different promoter-constructs by one-way ANOVA and post-hoc test. Analyzing the ct values is more accurate than transforming them (x-fold compared to sample xyz).

K.

-kr├╝melmonster-