I am trying to identify 2 most stable reference genes to be used in qRT-PCR. I am starting with 10 genes. After real time PCR it is observed that 18s gene shows most stable Ct values through out different tissue samples. After that CAC and ACT. According to BESTKEEPER analysis, 18s and CAC are the 2 most stable. But GeNorm, NormFinder and delta-Ct method in RefFinder is refering eIF4a as the best one, but the Ct values of this gene are not consistent ranging from 22 to 26. Also according to last 3 softwares, 18s is the worst stable gene. Which one should I believe? I can see 18s Ct value stable at 25 in different tissues.
Better to use several genes to get more accurate housekeeping values
What is your RNA input and are the samples equally loaded, i.e. each sample has 100ng input? It is interesting to note that your 18s is not so abundant (25 Cts), you may be using low input?
Can you show us a screenshot of the raw PCR curves data and the stability data.
Each software uses it's own algorithm to calculate stability. I found this paper: Selection and Validation of Reference Genes for Gene Expression Analysis in Switchgrass (Panicum virgatum) Using Quantitative Real-Time RT-PCR by Gimeno et. al., to be particularly helpful in understanding that although different software programs choose different combinations of reference genes as the most stable, the final analysis of the data remains the same. Common qPCR practice is to use more than one (preferably 3) reference genes to normalize your data. I would trust any combination of genes the different algorithms choose for you, as long as you have done your due diligence to give it enough samples to accurately pinpoint the most stable combination.
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