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ddCT issue: no endogenous expression in control.

Real-time PCR ddct

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#1 mohsen

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Posted 13 April 2012 - 02:23 AM

Hi

I have a question about ddCT method. If there's no expression of the target gene in the control sample, what value shall I use as Ct to do the calculations? For example, I'm transfecting the X cell line with an empty vector (treatment A) and also the vector containing the gene Y (treatment B). The X cell line does not express the protein Y endogenously. In this case I will have Ct values for house keeping gene in both treatments (A and B), Ct for the Y gene in treatment B, but no Ct value for the Y gene in the treatment A. What do you think shall I do? Someone told me I will have to put the number of the cycles of the protocol as Ct, but it does not make sense to me.

Thank you for the advise in advanced
Mohsen

#2 Trof

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Posted 13 April 2012 - 05:09 AM

Idealy use a different control. You cannot compare two things when one of the things doesn't give a result.
You can substitute the negative result for the detection-limit-Ct+1, what would probably someone interpret as number of cycles, but that brings many problems. Because in one setting you would have like 1000000x fold overexpression, and when you double the template amount and the control would be still negative, the ratio would now be different. But generaly you can count to the detection limit+1 and say "it's at least .... times more".

But the question is, if it really matters to have a real ratio, you can just say that control sample didn't have any expression but you sample did. If it trully didn't have any expression (not only not detectable) it would be illogical to try counting how much is something bigger than zero.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 mohsen

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Posted 16 April 2012 - 02:02 AM

Idealy use a different control. You cannot compare two things when one of the things doesn't give a result.
You can substitute the negative result for the detection-limit-Ct+1, what would probably someone interpret as number of cycles, but that brings many problems. Because in one setting you would have like 1000000x fold overexpression, and when you double the template amount and the control would be still negative, the ratio would now be different. But generaly you can count to the detection limit+1 and say "it's at least .... times more".

But the question is, if it really matters to have a real ratio, you can just say that control sample didn't have any expression but you sample did. If it trully didn't have any expression (not only not detectable) it would be illogical to try counting how much is something bigger than zero.


Actually I could not use another control, since we are studying some viral miRNAs, so we must use a cell line that is not infected with virus, i.e. no viral miRNA expression.

The point is just like you said, it does not make any sense to say how fold the expression is higher than zero. But at the end, I must present this as graphs, so there's a need to have a number. To me this is a downside of ddCT method.

I did not understood what you mean by detection-limit-Ct+1. Could you explain it a little more please?

Thank for the comment.
Mohsen

#4 Trof

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Posted 18 April 2012 - 01:51 AM

I did not understood what you mean by detection-limit-Ct+1. Could you explain it a little more please?

I heard it on some seminar, that this is a method how to quantitate negative results. Since number of cycles can be increased just by setting, using the muber of overal cycles as Ct value for negative sample doesn't have sense. So first you have to find detection limit, some definitions of this are in this example. As this is last Ct actually detectable with a sample, you must then add +1 to it for representation of a negative result. It some kind of mathematic tweek or something.
People speaking on the seminar were statisticians, and they were using this approach regulary in large amounts of data, when they simply needed a number. But I wasn't able to find any paper describing or explaining this in detail.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 mohsen

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Posted 01 May 2012 - 10:27 AM

Thank you so much. I'll read it.





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