hi

If you want determinate of primer dimer, you should better run the pcr product on 2% agarose gel.it seams no primer dimer on gel picture , also 180bp weak band Can be seen in lanes 2,4&6 of agarose gel picture .try to gradient PCR program for your PCR reactions.

good luck

Hi,

I agree with Reza. This might also be due to non optimal Primer sequences which might tend to produce dimers. In general it would be good to know the amplicon size here. How much Primers did you use? Sometimes a titration of the primer concentrations does also help

Best Sven

Ok, the first point I would make is that ActB is a questionable choice of reference gene: it appears to decline fairly consistently as myoblasts differentiate into myotubes (and you should ideally use multiple ref genes anyway).

Article Identification and Validation of Quantitative PCR Reference ...

The second point is that melt curve analysis must always be interpreted carefully, taking into account the full context of the reaction conditions.

For example, if your ActB NTC wells produce a melt peak identical to your actual cDNA samples, this may simply indicate you have a tiny, tiny amount of contamination in your PCR buffer: ActB is a highly expressed gene, so it wouldn't take much aerosol contamination to get ActB positive results in NTC wells. What really matters are the Cq values: if ActB gives you Cq values of 18 when you have template, but 32 when you do not, that is basically ok: you have a tiny (~1/16000)

bit of contamination, but it will not affect your actual measured results at all.

This also plays into cases where you have conditional melt peak behavior: Myh1 gives you two peaks in undifferentiated cells because you are simply measuring noise. Undifferentiated myoblasts do not express myosin heavy chains. The noise disappears as your cells differentiate, because now you are measuring actual transcription, not noise. This should be clearly reflected in the measured Cq values and the amplification traces.

The only real cause for concern is your MyoD, which should be robustly expressed in myoblasts and also in myotubes. if you are seeing two peaks here, and seeing them in NTC wells too, first check your Cq values: hopefully those for actual template are much lower than NTC.

Secondly, check how many cycles you are performing, and where in that number of cycles your NTC wells produce amplicons. PCR is an exponential process, so with enough cycles, you will amplify SOMETHING. Similar to ActB, above, if your NTC wells produce amplicons long after your template wells have saturated, the second aberrant peak in the myoD PCR may simply be late-appearing primer dimer. You can always try a run with fewer cycles, including just enough to amplify your myoD template wells to saturation, but not enough to obtain NTC amplification. The melt curves for these should not show the extra (low temp) peak (and if they do, redesign your primers).

Thank you @johnhildyard, @svenreister, and @rezahadavi for your informative responses on my qPCR question.

The primers used were 0.2uM (200nm), which was the recommended amount to use. Also, I was following a protocol that a previous student used for similar purpose which included 40 cycles. It was my first run.

After reading these responses, I carried out another qPCR using less cycles as well as a different qPCR kit, and I did not have any low temp peaks in any of my primers nor did I get amplification for my NTC. It was a lot of give and take and seeing what works. I also did a temperature-gradient qPCR to optimize the annealing temperatures before redesigning my primers, which also contributed to the success of this qPCR run.

Thank you guys! You have been very informative and helpful!

Hi, John Hildyard

What if the cycle numbers for GOI and HKG are different? Is it acceptable or not?

I used GADPH for HKG and PAF-R for GOI

I did 40 cycles for HKG (GAPDH), but the NTC always amplified around 26-32 cycles, even after changing all the master mix, water, and primers with new ones, preparing everything freshly, sterilizing all the tips, and using aseptic techniques.

So, I changed the cycle number for GAPDH to 30 cycles, but the NTC still amplified at 33 cycles.

For the GOI, I used PAF-R, and the samples amplified around 24-33 cycles, while the NTC did not amplify.

Does anyone know about this and can give me a solution?"

Let me know if you need any further modifications!

Ok, think about what your data is telling you:

if your NTC is amplifying for GAPDH but not PAF-R, despite these sharing all the same reagents/tips/etc EXCEPT the primers used, then...the GAPDH primers are the problem.

Probably they're forming primer dimers (if the melt curve for the NTC amplicon is ~70-75 degrees, then yeah: primer dimers).

The next question is...is this a problem? You don't list what your Cq values are for your actual samples, but if GAPDH gives Cq values of...eh, 18-20, then you're basically fine: contributions from primer dimers will never meaningfully affect your data, since they'll only start to emerge significantly after your _real_ signal has already been quantified.

Remember: qPCR measurements evaluate the CYCLE at which a reaction reaches some threshold. That's your number, and after that cycle nobody cares what happens to the reaction. Running the reaction for only 30 cycles isn't going to make any difference.

In other words:

Real sample>>PCR>>GAPDH amplified, Cq of 18>>subsequent primer dimers also amplified, nobody cares

NTC sample>>PCR>>no GAPDH, primer dimers eventually amplified>>Cq of 28

A Cq of 28 represents 1000-fold lower expression than a Cq of 18, so even if it's contamination/noise/primer dimers, it will at best affect your quantitation by 0.1%, which is substantially less than contributions from more general stuff like pipetting errors.

So: basically, what are your actual sample GAPDH values?

(for what it's worth, a Cq of 26 for primer dimers is QUITE low, and if you can, I would redesign those primers. A Cq of 32, on the other hand, is basically fine for most comparisons: below about cycle 30 you're dealing with countable numbers of molecules, so if your actual expression is down in the 30s it's probably not meaningful)

John Hildyard thank u for your informative response

the cq value samples for GADPH is around 13-15

and cq value for NTC is around 28-30

means, a cq 30 represents 32,000-fold lower expression than a cq of 15, so this affecting the quantification lower than 0.1%?

is basically fine?

Basically fine, yes.

I suspect you could also dilute your cDNA a LOT (maybe 1/10 or 1/20) and still get entirely useable quantitative data. It would also make the cDNA last longer. I typically dilute all my cDNA stocks (for qPCR) to 1/20.