Hi,
we are performing qRT-PCR assays, we did a triplicate under the same conditions and run the melt curve analysis. We obtained a single peak, but the height was different. Anybody knows why this is happening?
Thank you very much in advance.
Hello. In your figure the peaks are lying in almost similar temperature range while the only difference is their height. One probable explanation could be amount of your sample differs in your triplicates and this could possibly happen due to pipetting errors or possibly your samples evaporated (some liquid) during the qPCR run time due to the ambient temperature you are working probably the cap was not closed properly/tightly. Try to mix your samples after adding all the buffers and mix then spin down to properly mix it.
Hello,
I do agree with Harvey, it looks like a pipetting error within your triplicate. Could you please confirm that you do prepare one Per mix with your sample matrix and dispense it 3 times in three different wells of your PCR plate? Did you use manual or electronic pipettor? I would highly recommend electronic pipettor or liquid handling robot if your lab gets one. It will decrease both your Cq and melting curves variations.
Feel free to contact me if you need further info
Best.
Arnaud
Hi,
there are more possible explanations from my perspective and some options to rule out which might it be
1. Difference in Setup : How does the start fluorescence signal of the raw data look like? In addition how does the Cq value look like? If they are pretty similar this is not the explanation
2. Difference in PCR product yield. Although Cq values are similar position effects on the insturment or other external factors can influence the PCR product yield. Pls check if the melt curve height is similar. Also check End RFU values of your amlification plot (check raw data).
3. Difference due to algorithms. This is also a probable explanation, again check raw data and see how similar the reuslts are
In general this melt result is no reason to be concerned as the primary purpose of melt is to show presence of one peak at the same position which is clearly the case. Still check Cq values etc to see if your whole experiment is valid
Good luck
Sven
Dear Juan, dear colleagues, I agree with all suggestions, but I would like to add one more.
About different heigh of peks, it could bu due to different state of your wells (I mean plate and wells in your amplificator), after the thermoblock washing and dust clearing you probably reseive more uniform results. Moreover it could depends on DNA qantity bias because of pipetting.
For me it is look like normal results. Peak high is not so important if you can discriminate it. I say more, when we use simple Sybr (or similar) we can receive melt shifting within 1-2 degrees fro the same sequence. For more precision EvaGreen and anologues for HRM is available.
I hope that this information can help you. Good luck.
Thank you very much for your kind responses. We will take all of them into consideration in order to improve our results.
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