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With 40 pcr cycles, how relevant are samples with Ct's of 35-40?


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#1 wozzels

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Posted 10 March 2010 - 01:04 AM

Hi,

If i run 40 pcr cycles in a real-time pcr experiment, are Ct-values up to 40 relevant and meaningful, or should i discard results having a Ct of more than 35? And do blank samples have Ct-values equal to the max number of cycles, or not? Because if they do, unknown samples with high Ct-values may indicate the absence of DNA template right?
This is a question that is bothering me for quite some time now because I don't want to reject possible positive results but I also do not want to draw conclusions about samples which may not even contain my gene of interest.

Thank you guys in advance!
Wozzels

#2 ivanbio

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Posted 10 March 2010 - 01:35 AM

The relevance of a qPCR result is relative to the signal you obtain for a positive control sample. For example, if you have a positive with a Ct value of 30, then a sample with a reading of 35 may be relevant.

Think of it this way: assuming your assay is 100% efficient, then 6.6 PCR cycles are equal to 2 orders of magnitude. Another way of looking at this is: if you got say 1,000 copies of a gene equal to a Ct of 30, then a Ct of 36.6 is equal to 10 copies (2 orders of magnitude less, or 1% of your sample with a Ct of 30).

Most experts would agree that a background signal of 1% is minimal and thus considered to be not relevant (i.e. if your NTC shows up 6.6 PCR cycles later than your sample with the least amount of gene expression, then you can assume that your background noise is acceptable). I prefer to be even more strict and require a background noise of 0.1% (or 10 PCR cycles), but 6.6 PCR cycles for most intended purposes is good enough.

Hope this helps.

Ivan
Carlsbad, CA

#3 wozzels

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Posted 10 March 2010 - 02:58 AM

The relevance of a qPCR result is relative to the signal you obtain for a positive control sample. For example, if you have a positive with a Ct value of 30, then a sample with a reading of 35 may be relevant.

Think of it this way: assuming your assay is 100% efficient, then 6.6 PCR cycles are equal to 2 orders of magnitude. Another way of looking at this is: if you got say 1,000 copies of a gene equal to a Ct of 30, then a Ct of 36.6 is equal to 10 copies (2 orders of magnitude less, or 1% of your sample with a Ct of 30).

Most experts would agree that a background signal of 1% is minimal and thus considered to be not relevant (i.e. if your NTC shows up 6.6 PCR cycles later than your sample with the least amount of gene expression, then you can assume that your background noise is acceptable). I prefer to be even more strict and require a background noise of 0.1% (or 10 PCR cycles), but 6.6 PCR cycles for most intended purposes is good enough.

Hope this helps.


I understand, but what if your NTC gives a 'undetermined'-value instead of a real Ct?
Can you still verify the plausability of your samples in this case? (because this way you have no idea of how strong the background signal is)

Greetings
Wozzels




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