I have conducted three replicates of samples using quantitative PCR. Each sample shares the same conditions, with the only variation being the cell passage. Although I obtained similar Cq values for my genes of interest across the three replicates, the Cq values for Actin (the reference gene) differ: for replicate 1, the Cq values range between 16-17; for replicate 2, between 17-18; and for replicate 3, between 21-22. Consequently, this variance leads to different fold changes for the studied genes (0.8, 0.7, and 1.3).
Is the disparity in Cq values for Actin acceptable? Can I average the three-fold changes? Or is it inappropriate to average values less than one with those exceeding one? How can I determine the optimal fold change for my results?
Is there correlation between cell passage and observed Cq values? Do you see a decreasing/increasing Cq trend with passage number increasing? I'm not sure if that's a cell being brought out of cryo, perhaps as the cell is recovering from cryo storage the transcript expression increases or even decreases depending on what that gene does. Did you run a gene stability expression analysis (geNorm, RefFinder, BestKeeper etc)? I often found that genes that are assumed to be relative in the literature are not necessarily stable in my experimental conditions.
Thank you, Péter Gyarmati and Can Justin Kiessling , for your insightful contributions.
Péter Gyarmati I've taken steps to normalize my RNA before conducting qPCR experiments. However, I'm uncertain whether the fluctuations observed in the Cq values of Actin are indicative of technical issues or genuine biological variation.
Can Justin Kiessling I've observed that cells with lower passage numbers exhibit lower Cq values for Actin (between 16-18), whereas those with higher passage number show slightly higher Cq values (between 20-21). These cells were maintained in culture for three weeks prior to my experiment.
I'm seeking advice on how to analyze or average my data in this scenario. Any suggestions would be greatly appreciated.
If you are using a reference gene for normalization purposes, I advise you conduct a reference gene study to assess the stability of your reference gene under your experimental conditions. Difference of 16-21 is a pretty big change for PCR, 5 cycles is 2**5 = 32 fold change. What if your gene of interest was not changing in expression constant at around 21 Ct for the three replicates, then it'd look like you have big differences in gene expression for replicate 1 & 2 when your reference gene is the one changing expression. Did you have a look what the Delta Ct is between your reference gene and target gene? Conducting a reference gene stability test is not difficult, you need about 5-6 reference genes (Ppia, Beta-actin, Gapdh, etc) and pick an analysis suite such as geNorm/RefFinder/BestKeeper/etc. Some thermal cyclers have this feature built-in. I'm not sure if you chose beta-actin because some colleague recommended it or you found it in a paper, I've found that in the PhD studies I conducted, some of the commonly used reference genes were not so stable in my samples.
Thank you all for your valuable contribution to addressing my inquiry.
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