My project is about detection of species DNA in feed sample. I get a strange melt curve result from a developed species detection assay. The assay was optimized with genomic DNA. Peak at Tm=74 is the target species, peak at Tm=80.5 is the internal control species. Attached is the melt curve analysis.
Positive control gave me two significant peaks. Some blind sample showed an expected peak at 80.5 while few of them gave me not only an expected peak at 80.5, but also an addition peak at Tm=77.0. I tested those blind sample again in separated tube (singleplex) and realized the extra peak is actually derived from internal control.
My question is, whether this duplex qpcr result is valid? Is the extra peak matters?
Please share me your thought. Any opinion is welcomed.
Hi @carin, you reminded me of my mistake in the post. In fact, I am working on the qpcr using genomic DNA as template. Is seems the assay is invalid to you ya! Thanks for sharing your precious opinion.
Interestingly, when I ran the same sample by using a higher concentration internal control primer, the extra peak disappeared. But I couldn't compromise this because adjustment of primer concentration again will make a problem to the optimized assay.
I hope finding a new target for internal control is not the only choice.
Dear Lee Lee,
Use uMelt to take a principle view of your PCR products and to arrange PCR products with enough Tm difference. Actually what kind of reporter fluorescent dye use your Master Mix?
Also, if it is not problem please send me your PCR products sequences on [email protected]. I would like to investigate your seqs.
Kind regargds
Dear Dimitar,
Nice to hear from you~
Attached is the uMelt result. It showed a smooth dropping curve. In addition of GC content = 50%, I'd just expect that the dissociation curve will give me a single, sharp peak. And I got it when tested with known gDNA. Strange result was obtained when I tested on some blind samples.
The assay is SYBR green (reporter) based. ROX is the reference dye in mastermix.
And I am sorry I couldn't send you the sequence now Dimitar unless I get the permission from my boss. I hope you could understand my situation. However, feel free to ask and I will do my best to help to investigate the sequence.
Dear Lee Lee,
Please pay attention to the upper left corner of uMelt page - there you can see two buttons "Melting Curve" and "Derivative". Please select derivative to see peak/s.
No problem if can not send the sequences. I just have personal ideas for sequences investigation.
In case your MM is SYBR Green you should select sequences with minimal 8 degree Celsius difference between single peaks.
Also you should run every target in singleplex reaction before multiplexing. Run every target with different DNA concentration per reaction. To find appropriate Tm for your target vary the length of sequences.
Kind regards
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