I'm looking at Arg1 levels in untreated and treated macrophages. The different stimuli seem to have different effects on my macrophages, but I don't know how to calculate the fold difference so that I can present the results in a figure.
I got the following results in my qPCR (40 cycles):
untreated control Cт Mean Undetermined
treatment 1 Cт Mean 25,31
treatment 2 Cт Mean 34,52
treatment 3 Cт Mean 34,98
treatment 4 Cт Mean 35,53
treatment 5 Cт Mean 28,07
treatment 6 Cт Mean 35,64
Does anyone has an idea?
Thanks! :)
Ulrike
Hello Ulrike,
Are you familiar with the delta delta CT method? this method uses your control or untreated sample as a control and then you use a very straight forward method that calculates the "fold change" of your experimental sample comparing it with the control. However, although normally you use an untreated sample as a control, maybe your target its not expressed under those conditions. in this cases, you could use a positive sample (your positive control sample) as the reference material and then calculate the 2^-delta delta CT. If you would like a little more info please don`t hesitate to contact me. Here is the reference that probably you should read in this matter.
http://www.ncbi.nlm.nih.gov/pubmed/18546601
http://www.ncbi.nlm.nih.gov/pubmed/11846609
Best regards
No, you can't accurately calculate gene expression if your NT control is undetermined. As I surrogate, you may use Ct 40 for NT sample? This may not affect much of your results. However, ddCt will not work in this case, only dCt ( you may see my PNAS article in 2006). Did you measure Ct of housekeeping genes? What was RNA concentration and quality in your RT step? Human or mouse macrophages? Where did you get primers ( sequence)? Please try to repeate and I would be happy to help you to solve this problem.
Pi see that Julio suggested you the same. Good luck.
Thanks for your fast response and your help.
I'm working with mouse macrophages. We are using pre-designed QuantiTect Primer Assays from Qiagen and also the QuantiTect reverse transcriptase Kit and SYBR PCR Kit. The amplification efficiency of all primers was tested. I prepared a dilution series (0,001 ng - 50 ng cDNA) of a sample expressing Arg1 and four controls (Actb, Hmbs, Hprt, Tbp). The Arg1 primer assay has a different amplification efficiency than the four controls. For the experiment the two controls with the most similar amplification efficiency were chosen, in this case Hprt and Hmbs. For evaluation of the data an efficiency corrected calculation model will be used. We don't have much RNA, thus we only used 100 ng RNA for RT and 5 ng cDNA for qPCR.
- Badges
- Science topic