Question: qPCR gene expression confusion
gravatar for muha0185
12 months ago by
muha01850 wrote:

Hi I will be happy to get advise on this matter. This seems like a useful avenue to gain advise and more so since I am an undergraduate student who has a lot to learn.

So my project involves differentiating ES cells into a particular muscle cell, and this differentiating protocol takes 40 days. I performed qPCR to check if certain key markers are elevated. I collected RNA and performed cDNA synthesis on ES cells, day 20 and day 40 cells. In the first run, i ran day 20 and ES (control) in one qPCR plate, and obtained fold change values based on delta delta CT method. I then did the same for day 40 and ES (same control batch) in one run.

[delta][delta]Ct = [delta]Ct,sample - [delta]Ct,reference 2^-delta delta ct = fold change

Thing is 4 of my markers showed clear increase in expression. But one marker was downregulated instead, in both day 20 and day 40. This may seem confusing but turns out ES cells also express this marker so using ES as choice of control for this case seems like not so much of a good choice. Having said that, downregulation may not mean the gene was not expressed at all as this was relative qPCR, not absolute. So if i can show that day 40 expression is higher than in day 20 will prove my point. But as mentioned, i ran qPCR on different plates for day 20 and day 40 (although using same ES batch control), so i cant compare day 20 and day 40 fold change values? I proposed running day 20 and day 40 sample on the same qPCR plate to look at fold change comparing day 40 and day 20 directly but this is the reply i got from my PI. I spent hours trying to understand this but I could not wrap my head around it. PI is on vacation and doesnt respond to emails for a week. Hope to get advise from you guys!

"I see that the analysis you have performed has been done using expression by undifferentiated ES cells as the control. Another way of doing this is to compare each cell’s expression over the fold change from housekeeping genes. Am not sure if you did this first and then compared all the genes. Could you clarify? If not, you could try to compare thy1 expression for each stage with the house keeping genes alone first. If there is no change over time, then we definitely have a problem. We may need to test further with thy1 antibody before doing our panning."

rna-seq qpcr rna gene • 377 views
ADD COMMENTlink modified 12 months ago by ATpoint36k • written 12 months ago by muha01850

Thanks for such a long explanation! I would suggest adding your question in a single line at the top of the post, and go more in depth later on. It's quite a long text for us to quickly see if we can help you yes or no.

ADD REPLYlink written 12 months ago by WouterDeCoster44k
gravatar for ATpoint
12 months ago by
ATpoint36k wrote:

Hi muha0185,

first you should always normalize the expression of the target gene to an internal housekeeping gene. If you do not do that your results are meaningless as any changes in expression could simply be caused by loading more or fewer amounts of cDNA. The value you get from this is then used for delta/deltaCT between the two conditions. I do not think that the day 20 and 40 samples have to be on the same plate as you compare to the same control sample especially if you use the same reagents, pipetts, machine for everything.

ADD COMMENTlink written 12 months ago by ATpoint36k

I did do a housekeeping internal control.

delta Ct = Ct fav gene - CT actin [delta][delta]Ct = [delta]Ct,sample - [delta]Ct,reference 2^-delta delta ct = fold change

ADD REPLYlink written 12 months ago by muha01850

So what is the question then? Please shorten it, it is difficult to get the question in this wall of text.

ADD REPLYlink written 12 months ago by ATpoint36k
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