I am a master student doing a project where I am investigating circulating miRNA profiles. I did a mock RNA extraction, i.e. treated RNase free water as if it was plasma, including a subsequent reverse transcription and qPCR. I am using Exiqon's kit for miRNA qPCR analysis where you buy a ready-to-use-plate where all the primers, for spike-ins and 179 separate miRNAs, are already present in separate wells. When I ran my mock RNA sample I get amplification in some wells (i.e. certain miRNAs but not all) with a melting curve with no peak. Amplification in this sample would suggest contamination, however, the sample is treated with DNase during RNA extraction. I am nonetheless making a new mock extraction, hopefully without contamination, to see if I get other results. Thoughts and comments about the amplification and contamination is appreciated but what confuses me is the melting curve, how can I have amplification but no peak in the melting curve (See images included)?
The Ct is really high, so it might be the signal of an unspecific product. If this has a Tm below 60°C (what can happen), then you would not see it melting in a melting curve starting at 60°C.
If you used sequence specific detection like hydrolysis probes ("Taqman"), you would not expect getting melting curves anyway.
Have you checked the products on a gel?
What about positive and negative controls?
What about qPCR curve, do you see it?
What method do you use: sybr or TaqMAN.
The worst suspection, is that what you think is "your" product actually is non - specific amplification.
In my opinion you have non specific amplificaton. Therefore you see something in the amplification curve, but no specific peak in the melting curve. Your amplification curve looks like unspecific. There is no clear signal but after 30-35 a unspecific product seems to be formed and is the amplified. Those can be primer dimers for example.
I would suggest to run the PCR with any kind of positive control to see that your PCR settings are working in general. If this is fine then you should focus on your sample. Maybe your sample is empty?
The Ct is really high, so it might be the signal of an unspecific product. If this has a Tm below 60°C (what can happen), then you would not see it melting in a melting curve starting at 60°C.
If you used sequence specific detection like hydrolysis probes ("Taqman"), you would not expect getting melting curves anyway.
Have you checked the products on a gel?
What about positive and negative controls?
Thank you so much for your answers. I am using SYBR, and the negative control show no amplification and the positive controls give about the same Ct as on the 12 plates I've run prior to this one. This whole sample is a negative control and *should* be empty, containg no cDNA except for the spiked-in c. elegans cDNA (My samples are human plasma), which is why I was expecting no amplification in anything but the positive control.
We are currently not running our pcr product on a gel. From what I understand, running miRNAs on a gel is complicated to begin with because of their size, but is further complicated by the extremely low concentrations of miRNA in plasma, and I rarely see Cts below 30. Also, I'm running 10 ul reactions of two samples/technical replicates in a 384 well plate, that is a lot of gels for one plate, and I'm running a plate per day.
Hello Debora
Just Check the Reaction on 2% Agarose Gel with a DNA marker (100 bp Ladder), and check the size of the band (if visible) . u will get the clear whether it is amplicon of ur interest or simply a primer dimer . And plz note that primer dimer shows melting temperature below 80 degree celsius
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