my work on blaOXA genes in Acinetobacter and i want to use the method Delta Delta Ct for measurement the gene expression by using quantitative RT PCR assay , is this method give Certified results and is there another methods?what about the statistics for ΔΔCt if I use triplicate samples?
∆∆Ct is largely considered the best way to analyze gene expression results. Use it.
Regarding triplicate samples, that will depend on what you mean by triplicate. If you are talking about technical replicates, then average them and perform the analysis to result in a single biological replicate. If you are talking about biological replicates, then keep them separate, do the ∆∆Ct, then calculate statistical significance. Your statistical tests will depend on the nature of your sample collection.
Thanks,Dr. Jordan R. Yaron, yes I mean the technical replicates. The reason one performs these replicates is to control for the validity of the method. you advise me for specific statistical test suitable for gene expression ?
ANOVA or a t-test would do. I would highly recommend to read
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)
method. Nat Protoc. 2008;3(6):1101-8. PubMed PMID: 18546601.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001
Dec;25(4):402-8. PubMed PMID: 11846609.
∆∆Ct is the or Semi-quantitative-Real-time PCR. You may want to explore absolute quantification as well. In this method you have have a standard curve with known amounts of target DNA. This method will allow a PCR to PCR comparison.
Thanks Dr.Navneet Ammal Kaidery,
I appreciate your recommendation with my best rgards
no stats in the DDct method itself, stats will come when analysis samples and as it has been said with almost 3 replicates per samples, and really good designed primers for PCR to prevent from mis-amplification.
fred
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