Hi everyone,
I have been working on real-time quite alot. Recently, I performed an experiment with 3 repeats. For me, repeat means doing same experiment again, but at different time. So, I did real-time PCR separately for every repeat.
In my experiment, I was determining the expression of HSP90 while using Beta-Actin as internal control. Then, using 2^(ddCt) method, I was determining the fold change between my control and treatment.
Ct values of my repeats for Actin (22.5, 24.6 and 20.94) and ct values for my HSP90 are (26.4, 25.66 and 23.57) for my controls.
and
Ct values of my repeats for Acting (24.338, 26.155, 23.191) and ct values for my HSP90 are (28.48, 29.077 and 25.349) for my treatment
Keeping this thing in mind... how can I pool my data? and how can I be sure that the difference between my test and control is significant or not? I shall be looking for your response.
Regards
In my opinion, 3 repeats are designed to control random variation caused by pipeting or instrument. I suggest you repeat the experiment, with 3 repeats of each sample. Besides, I guess you've used a sybrgreen method, and weahter you have tested the specificity of the primers accurately?
Thank you Fred for your response,
Following things were done in each real-Time PCR...
1. Each real-time PCR was carried out in Triplicates. So, the values per experiment shown above are average of the technical replicates.
2. I performed standard PCR for every Primer and checked the size of the products and non-specific amplifications. I found nothing such. Second, I performed the melt curve analysis in the real-time for each and every reaction. So, I have without doubt pure amplifications...
3. Yeah, I am using cybrGreen chemistry to perform my real-time PCR..
I'd simply calculate the dCt values of the paired measurements as usual. This should eliminate any between-run differences. Using some ANCOVA model or a mixed-effects model is not promising because of the low number of replicates.
Have you checked that you have the same threshold value for each target in each experiment?
When I get a new set of primers I do a standard curve on a serial dilution of cDNA to obtain the efficiency of the primers and the threshold value that I set for the efficiency is the one I use throughout all my experiments.
qPCR is just a tricky technique when it comes to obtaining good biological triplicates, because it is so sensitive.
Hope it will work out!
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