Hello to all dear students, researchers and professors all around the world. I have a question about Real Time PCR (Rotor gene Q -corbet): I use “TaqMan Probe” for detection of KRAS gene mutation; my wild type probe is “Hex” and my mutant probe is “Fam”. I have 2 main samples, KRAS positive and KRAS negative (as control) + NTC sample. After finishing the process, I see the amplification curve of wild type and mutant at the same time in both canals (yellow and green). Technically, I must observe each curve in related canal, but it’s not like this (I'm not suspicious of the "Heterozygous" or "cross contamination" scenario!). I will be grateful if you help me. Thank you for reading my post. Please feel free to make contact with me.
What exactly is the problem here, did you get unexpected signal in the KRAS negative channel for the FAM channel? Amplification curves with samples and what you'd expect in each channel (FAM +, HEX +) would be useful for each sample.
Can Kiessling
My probe for mutant type is FAM (it's green) and my probe for wild type is HEX (it's yellow). So when I try to see the green channel, I must see just the mutant amplification curve and when I try to see the yellow channel, I must see just the wild type amplification curve. But I see both curves in both channels!!!
i rule out these scenarios:
1) Cross contamination: because my NTC is clean and OK
2)Heterozygote condition: it's rare that both samples be heterozygote at the same time and also, my 2 samples have 2 different origins; KRAS + is DNA of a cancer patient that is extracted from his FFPE sample and KRAS - is DNA that extracted from the WBC of a normal person.
3)Spontaneous hydrolyzation of probes: i don't see any jagged amplification curve in my NTC
I'm just suspicious about 2 scenarios; my wild type and mutant type samples have a difference in only 1 nucleotide (as pathogenic point mutation) and because of that, maybe my probes don't have enough specificity or good design.
OR
Maybe it's normal to see both curves in both channels (because of samples difference just in 1 nucleotide), but with 1 exception! CT number!!! means that, I must see mutant curve 1 or 2 or 3 CT sooner than wild type curve in green channels and vice versa!!! And then, rule out irrelevant curve with delta CT!
I don't know how to figure out my problem! At least, now! :-)
I would guess one nucleotide difference is just not enough to affect Ct values, as this will really come down to the Tm of the surrounding nucleotides. It will also depend on what polymerase (with what error rate/precision) you use.
Article Evaluation of the impact of single nucleotide polymorphisms ...
Péter Gyarmati
thanks to you for sending that article.
you make me happy if you share your idea about this problem. I hope to fix it so fast. thanks gain.
"Heterozygote condition: it's rare that both samples be heterozygote at the same time and also, my 2 samples have 2 different origins; KRAS + is DNA of a cancer patient that is extracted from his FFPE sample and KRAS - is DNA that extracted from the WBC of a normal person"
If you are using clinical samples, I wouldn't agree that a KRAS positive (G12X, G13X, etc) would only be FAM positive - I've worked in oncology for 5 years and I have never seen a homozygous KRAS positive allele using Next Generation Sequencing data. Plus, with PCR detection approaches you generally want a wild-type control or an external control to be positive at all times as a reaction positive control. So your KRAS positive sample should also be HEX positive at all times because wild-type allele is present in all samples. One other thing that I could think of is the FAM-HEX crosstalk between these channels but FAM-HEX combination is widely adopted and not a known source of noisy combination to my knowlodge (see pg. 9 3.2.2 of https://gmo-crl.jrc.ec.europa.eu/doc/KJNA30708ENN.en.pdf). By the way, did you try just adding 1 of the probes to your KRAS positive samples, if you suspect bleedthrough from FAM to HEX, then you'd see this if you added just 1 of the probes and take a read. See figure 4 of https://www.biosearchtech.com/support/education/multiplex-qpcr
That being said, a strong FAM positivity can cross-talk into HEX channel
(see pg 2 of https://www.qiagen.com/us/resources/download.aspx?id=df035923-b6a4-49a8-9cb4-8b9edbafce61&lang=en)
Can Kiessling
Dear Can, thanks to you for your help and documents that you tried to share with me... I will try to read these...
I will be in touch with you. god bless you.
If you want to rule out channel cross-talk between FAM and HEX, just do this in separate PCR tubes:
1) FAM probe only in KRAS positive.
2) HEX probe only in KRAS positive.
3) FAM probe only in KRAS negative.
4) HEX probe only in KRAS negative.
5) FAM probe only NTC
6) HEX probe only NTC
That'll give you an idea whether there is "bleedthrough" with these 2 channels. If any of the samples is double positive, then that's your indication that there is some cross-talk between these channels. If there is no cross-talk, then looking into specificity might make sense as the next point. Are you confident in your TaqMan probe to discern a sequence variant?
Can Kiessling
Dear Can, thank you again for all of your help and attention. God bless you. You are a good friend.
It seems like you are using RotorGene, have a look at the manual for your model and see if the FAM + HEX duplex combination is recommended. I checked for the 3000 and 6000 models and it seems OK for duplex. See page 17 and 19:
https://www.qiagen.com/us/resources/download.aspx?id=e7ca84fe-de1a-4b33-8073-72ebd0d10ebd&lang=EN&ver=1
Hello dear Can, I sincerely appreciate your support. God bless you.
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