Posted 11 February 2013 - 03:19 AM
I am trying to analyse gene expression over a time period. I used the delta delta Ct relative quantification method for analysis. My endogenous control is Actin and my calibrator sample is the cells obtained from the first time point (Undifferentiated cells).
After completing the first round of experiments, it is evident that my gene of interest is not expressed in my calibrator sample (time 0: undiff cells). Therefore I am unable to calculate the fold changes in gene expression relative to this time-point. The endogenous gene was amplified in my samples, but the gene of interest was not. Moreover, my no template control and negative control did not show any amplification.
Ultimately I would like to show that my gene of interest increases over time. Is there another method that any of you can recommend I use to analyse my data?
Posted 11 February 2013 - 02:01 PM
Posted 11 February 2013 - 02:44 PM
Do you know if this is possible?
Posted 11 February 2013 - 04:40 PM
Expression of your GOI within a plasmid should be relatively consistent. It should not decrease or increase over time. That is why (basically) all vectors contain strong promoters before the MCS. You should also keep in mind that when you are running a RT-PCR you are looking at a snapshot of the cell. You are looking at all the RNA that is present at that point in time. If you were to isolate 10 RNA samples from the same cell line at the same point, expression of your plasmid+GOI would vary. It is important to determine if your plasmid+GOI is being expressed via RT-PCR, but I would personally stick to using primers that cannot distinguish between your endogenous and exogenous gene. Only because it will give you a better understanding of expression change at the RNA level.
Did that answer your question?
Edit: I can't write a cohesive sentence.
Edited by jerryshelly1, 11 February 2013 - 04:45 PM.
Posted 12 February 2013 - 03:57 PM
Thanks for your replies.
I am trying to use the relative quantification method (not absolute). Hence, I believe it is not necessary for me to create a plasmid.
Therefore my question is, how to calculate fold change in gene expression if your gene is not expressed in your calibrator sample? I think I may just have to choose another calibrator sample and calculate the fold changes, and merely note that my undifferentiated cells did not express my gene of interest.
Also does anyone know how to calculate delta standard deviations for fold changes?