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Problem with real time PCR


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As I am from South Africa, real time PCR is all new to me. I was very excited to use this new technology on my masters project but with great disapointment. I'm looking at polymorphisms in rare gene so a positive control was not possible at first. Because of this the automatic allelic discrimination software doesn't call work as well.


So the big problem is manual genotype calling using the amplification plot. Now this was a big joke. Nowhere can you find info on how to do it. I contacted three reps which included Bio-Rad, Applied Bio and Roche. According to all of them when you get two signals (FAM and VIC) no matter what the amplification of each then you can call it a heterozygote. Is this true.


I'm really disappionted with the technology. Even when we tried HRM for diagnotic work on breast cancer the results were inconclusive. Can anyone please help with this.



Edited by Sue-Rica
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I have never done real-time quantitative PCR but I have studied papers that use it. From what I've seen I'm not impressed, and the results have been inconclusive and the conclusions drawn from them unconvincing.


I'll update this with more specifics later when I can find an example.

Edited by Greippi
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Real-time PCR covers a wide number of application and it works excellent for some, lousy for others.

But first of all, no bioanalytical technique is 100% accurate (depending on what is determined as accurate, i.e. accurate detection of a certain molecule is not equivalent to an accurate determination of a state of the organisms).


That being said a problem with the use of real-time for quantitative analyses is its semi-quantitative nature, coupled with the exponential signal increase (i.e. small inaccuracies in the method can lead to large differences).


That is why controls are often crucial. The use of the target sequence (e.g. synthesized oligos or cloned constructs) can really help in normalization.


Depending on what you really need (i.e. quantitative vs qualitative data, throughput etc.) a traditional PCR can lead to more stable and reproducible results.

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Thank you for replying. My big problem is the Ct values. In theory if you have 2 Ct values one for fam:20.12 and one for vic:20.98 then it is a heterozygote. But where is the cut off Ct value for heterozygotes? We made it a diffirence of +- 1 Ct.


If you have an amplification plot with fam having a higher RFU than Vic but the Ct value of vic is higher is it then homozygous for fam or vic?



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There may be a little bit of confusion here. Just to clarify, the ct value indicates when a certain amount of product has been produced (using the first derivative of the curve). I.e. it is dependent on the efficiency of the individual PCR reaction. As such there is no absolute cut-off value whatsoever. If your PCR mix is optimal (in terms of purity and perfect ration of primer, target, polymerase, etc), you will have the lowest value, if the situation is suboptimal, it will take more cycles, the curve has a different slope and hence, the ct changes.

What it really tells you, however, is that if the CT value is not too high (at very high cycles unspecific signals occur), you got a product. This qualitative analysis is similar to standard PCR and gel-based detection.

Using calibration curves you can then use the CT value to assess, semi-quantitatively, the initial target concentration. This is the basic use of qPCR.

Note, that you cannot easily cross-compare PCRs with different primers, as all quantitative differences can be due to difference in primer efficiency (i.e. you cannot easily infer abundance based on CT values for different alleles.


Now regarding your example, if you got two significant signals, regardless whether in gel or using real-time, it means that two allels are present in your samples. And hence, it is heterozygous. If it was homozygous only one, or the other signal would be apparent (in the same sample, of course).

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So what you mean is that it doesn't matter what the amplification (RFU) or the Ct value is if there are two curves then it is heterozygous regardless of the intensities.


Let say orange represents allele 1 and blue repersents allele 2. On the amplification plot (where you see curves) there are both orange and blue curves but with there two examples:


1: Orange and blue are on top of each other meaning a very clear heterozygote and no difference or space between the two curves, the Ct and RFU's are exactly the same


2: The orange curve is at top and the blue curve just above the threshold, meaning the difference between the two curve are huge


Is number two also a heterozygote


Example pictures of number 1 and 2



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Well let us interpret the curves in more detail. First, in order to differentiate between the two alleles I presume that different primers were used. That alone will result in differences in amplfiication efficency and thus render a direct comparison between orange and blue irrelevant. I.e. you generally can only compare CTs of orange to orange (one set of primers) and blue to blue (the other set) without further normalization.


However, one tricky bit is determining the threshold, i.e. where you determine the CT. Note that in your two plots the indicated threshold would result in very close CTs, however in plot 2 the absolute signal in the blue curve is much lower.

The shape of the blue curve in plot 2 indicates that the efficiency of the PCR is very low. One key element here is the threshold. It should be:

1) way over the background noise of the initial cycles to ensure detections and

2) be in the exponential part of the amplification curve


Looking at plot 2 it appears to me that the threshold is very close to the initial noise (i.e. early amplification cycles where there should be no meaningful signal) and the exponential part is hardly visible. Based on that I would conclude that the threshold has to be higher and no proper CT can be derived from the blue curve. In other words, the curve appears not to be indicative of a proper product. The observed signal could be easily background (again, note the hump at the beginning) or unspecific amplification.

Either the sample contained no blue allele, or the PCR simply failed for technical reasons. To be certain I would have to see the actual values, of course. The melting curve could give hints whether unspecific amplification may have occurred.

Of course, the PCR could simply be super-inefficient for the blue primer pair, but looking at blue in plot 1 reveals that the curve can look very similar to the orange one (assuming in plot 1 the same sample was used).

So based on the curve shape and assuming the PCRs worked fine, my assessment would be sample1: heterocygote, sample 2: homocygote.

Note that the CT did not play a role (provided it is not something like cycle 30 or above, depending on the polymerase).

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