What can be the cause for different results in conventional PCR and real-time PCR?

In addition to my first answer,

Maybe one of the compounds, which you use in the qPCR reaction and not in the conventional PCR, is responsible for the inhibition of the amplification reaction by the polymerase and this in the case where you analyze by qPCR.

So to be sure, check by agarose gel and see if the amplification product is present.

Nikolay Klyashtornyy

There are several reasons why conventional PCR and real-time PCR (qPCR) can yield different results, even when using the same PCR parameters and primers. Here are some possible explanations:

Sensitivity: qPCR is generally considered to be more sensitive than conventional PCR because it can detect and quantify PCR products in real-time as they accumulate. Conventional PCR relies on endpoint detection, which may be less sensitive, especially if the PCR product is present at low levels.

Contamination: Real-time PCR is more susceptible to contamination because it involves handling of PCR products in real-time. Therefore, it is important to use appropriate precautions and controls to prevent contamination. Contamination can lead to false-positive results in conventional PCR, while qPCR may be able to detect and exclude such results.

PCR inhibitors: Real-time PCR is more sensitive to PCR inhibitors that may be present in the sample, such as hemoglobin or other substances that can co-purify with DNA. If present, such inhibitors can affect the efficiency of PCR amplification, leading to false-negative results in qPCR.

Differences in primer specificity: Although the same primers are used in both conventional and real-time PCR, the specificity of the primers may differ between the two methods. This can lead to differences in PCR product yield, with one method producing more non-specific products than the other.

Differences in reaction conditions: Although the same PCR parameters are used, slight differences in reaction conditions, such as the annealing temperature, extension time, or MgCl2 concentration, may affect the efficiency of PCR amplification and lead to different results.

Several factors can contribute to different results in conventional PCR and real-time PCR, including differences in sensitivity, contamination, PCR inhibitors, primer specificity, and reaction conditions. It may be helpful to optimize the PCR conditions and controls to minimize these sources of variation and improve the accuracy and reproducibility of the results.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

Danilo Reis Gonçalves

Thanks for the answers.

I suspect the presence of inhibitors in the DNA - extracted from soil samples (ex. humic acids), might be negatively affecting the qPCR fluorescense (I'm using sybr green). But the same does not happen with conventional PCR. I've found some papers in which they included BSA (bovine serum albumin) and it seems to improve it.

I'll run a gel with the qPCR products to see whether there was amplification.

Nikolay Klyashtornyy

You're welcome!

It's possible that inhibitors present in the DNA extracted from soil samples, such as humic acids, could be affecting the qPCR fluorescence. BSA can act as a non-specific DNA-binding protein and may help to reduce the effects of inhibitors on qPCR reactions. Additionally, BSA can help to stabilize the Taq polymerase during the amplification reaction, which may also improve the efficiency of the reaction.

Running a gel with the qPCR products is a good way to determine if amplification occurred. If amplification did occur, the product should be visible on the gel as a band of the expected size. If amplification did not occur, the gel should be negative. Keep in mind that a negative result on the gel could be due to factors such as low template DNA concentration or suboptimal PCR conditions, in addition to the presence of inhibitors.

Katie A S Burnette

The answer could also be as simple as the Sybrgreen has degraded/expired. It's sensitive to light and to freeze-thaw cycles. Most folks create "single experiment" aliquots to thaw & use just once.

I know it sounds counter-intuitive, but you might need to dilute your DNA samples 1:10 or 1:100. That dilutes out inhibitors, but leaves enough DNA for the PCR to still work.

As Nikolay Klyashtornyy said, try running out some samples on an agarose gel.

Good luck!

E. Jordán Muñoz-Adalia

Hello Danilo,

It could be interesting to check the melt curve of sybrgreen provided by your qPCR thermalcycler. There you could check if everything is going fine with your staining.

As other members mentioned, contaminations could be playing an important role thus creating "false-negative" results.

In addition, check the size of your expected amplicons. If they are so long it is possible qPCR polymerase cannot copy the complete length of your target region thus reducing the yield of the PCR or even resulting in "no amplification". Usually, we use amplicons with 300-500 bp in length for end-point PCR whereas in qPCR we usually work with

How can I measure MMP-1 protein activity or protein level from liver tissue?

Binary Logistic Generalized Linear Mixed model in SPSS. Is it doable?

Looking for Materials with high thermal expansion coefficient and high temperature resistance?

Guidelines for survey (question type) analysis?

What are the values needed for gabor filter's parameter for cifar10 data?

Can we block unwanted images during the x-ray prediction?

No title

What techniques can I use to determine the miscibility of polymer blends?

Sample dilution for quantitative analysis using ELISA?

The procedure compute?

What is your perception on using visual analysis or quantitative approaches to evaluate data from single-case research designs (n of 1 trials)?

How do I increase the yield of my PCR product?

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

How to address this issue in the analysis of Real-Time PCR results?

No title

MiRNA qpcr problem?

Runs of nucleotides in Sanger sequencing?

Can thermal cycler machines be problematic during PCR?

Calculating volume of DNA required from DNA concentration?

Has anyone used the Illumina ITS1 primer sets?