I have a very good setup for a real-time PCR assay using hydrolysis probes (FAM, they are not actually TaqMan but ordered from IDT instead). I achieved an efficiency of 97% and a sensitivity up to 5 femtograms of DNA, so I am very happy about the performance.
The point is that testing for specificity, my non-template controls do not produce any amplification while closely related bacteria, which I know for sure that do not have the target gene I am amplifying, produce fluorescence signal in very late cycles.
In this case, using 5ng of DNA template, my target organism amplifies about cycle 18-19 while closely related bacteria amplifies always in cycles above 36. Apart from NTC, I also added some reactions with 5ng of human DNA and did not produce any amplification either.
I know the difference between my target and non-target bacteria is huge, and I am not particularly concerned about this non-specific fluorescence.
I just want to understand what kind of non-specific interaction or by-product can produce fluorescence signal in a RT-PCR assay using hydrolysis probes, since in order to do that, that probes need to be hydrolyzed.
NOTE_1: I already analyzed primers and closely related bacteria sequences and, supposedly, my primers are not unintentionally amplifying any other region nor any combination of primer-probe seems to be capable of yield linear amplification.
NOTE_2: reactions are run in a Roche LC480 and Cps may seem too high since they are calculated using the second derivative maximum,
Dr, Bhowmick, are you getting similar results? In that case maybe we could discuss our results and try to elucidate what's going on. I'm actually going to perform some other experiments to know how the concentration of different non-target templates affects to this non-specific signal.
Hi Galo.
I also have the similar problem. I actually used the Taqman probe for the analysis of my GOI (NPM-ALK) and the reference gene. My negative control cell line had the amplification at about 33-34 cycle. And, my negative control (no cDNA) didn't have any amplification.
I will be repeating the experiment this week just to make sure that it wasn't an artifact. But, did you find any solution regarding the problem?
Dear Geeta, I repeated the experiments but this time I run an electrophoresis gel with qPCR products. It turned out that negative samples were actually amplifying, so non-specific interactions was not the problem. I repeated in silico analyses and I am quite sure those samples didn't have that gene I was targeting, so probably I got my samples contaminated at some point of the experiment. I am now growing and extracting DNA for those species again, and I am going to use new reagents for every single step, from extracting the DNA to qPCR, trying to assess if it actually was contamination. If I got again this amplification, then I will sequence the products by Sanger.
I will update this thread whatever results will be.
PD: Did you check possible amplification in other regions with primer-BLAST and possible primer-probe hydrolysis?
Try also to separate you positive and negative samples as much as possible, for example loading in opposite corners of the plate. Also you could first load negative samples, seal the plate with the sealing foil, and load then the positive ones by moving upwards the sealing foil just enough to give you space to load the samples so the negatives remain covered in this process. That depends on how many samples you have.
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