do you get negative results from your zero sample control. One possibility for these results is reagent contamination with pcr amplimer from the heterozygote if that is the result that you are getting.

We ran two no template controls (NTCs) with each assay, and we didn't get fluorescence from the VIC and FAM probes, so we don't believe contamination to be the issue. We are concerned we are misunderstanding how to interpret the results from such assays and maybe this is hindering our analysis.

If you're using brain tissue it may be that you have to perform a clean-up on your template. Brain tissue is very fatty and can often require adjuvant addition to the PCR reaction (DMSO or Betaine) to ensure reactions are successful or require you to first clean-up your sample.

Try running your PCR product on a gel before performing allelic discrimination to ensure you are getting amplification as well. If you don't see bands it means the PCR isn't working and it's not a probe issue. You need to optimize. This could mean (1) diluting your template, (2) adding an adjuvant, (3) cleaning up your template first, (4) MgCl titrations, or (5) gradient PCR to find the optimum annealing temperature for you experimental conditions

Dear Naomi,

I have experience with usage of ThermoFisher genotyping assays. But for me is not clear in which conditions you could not identify the genotype. Once E3E3 frequency is very high and all of your samples will aggregate in one cluster, the software will not assign any genotype, as the software needs to analyse a set of at least two different genotypes detected (this is how it works on ABI software, while, I expect, CFX Manager would do it differently).

So, what I did was to identify at least one sample of each of the genotypes to be used as genotyping controls and then set up the experiments including them.

From the other side, did you use single or double wells for each sample?

Are all of the samples of the same DNA content? DNA content was my troubleshooting issue which I solved by applying Dry DNA workflow. My experience showed 2 ng/mcl of DNA solution was fine.

How many samples do you place on the plate (or how many tubes do you use)? in some cases few samples generate such results.

I do not ask if you performed technical validation of the experience.

Besides, I use prePCR and cycle detection fluorescence in order to have hints whether there were changes during amplification.

If you would use ABI software, you could also track samples by ROX passive dye in correlation with another dyes, in order to observe pipetting errors or evaporation or another effects.

Please, let me know if you could solve the issue.

Kind regards,

"Test brain samples are supposed to be homozygous E3/E3 and we don't seem to be getting that."

Could you explain what results you are getting? Are you not getting a fluorescent signal at all, or do the samples look heterozygous, or is it something else? I don't know if you can upload pictures to this site, but if you can, would you upload the Quantification and Allelic Discrimination figures from the Bio-Rad CFX Manager software?

Sometimes the TaqMan probes will give a small off-target signal corresponding to the absent allele. If you are running only homozygous samples, the software might call them as heterozygous because it detects both signals, even if one signal is clearly dominant and the other signal is background.

Do you have any know heterozygous samples you can run? That may help you - and the software - assign the samples more accurately.

Hi, Angela, thanks for your help. I've attached the PDF containing the results of our most recent SNP assay. This time, the system was able to call all samples, and the results were as predicted/as they should be (E3/E3). I think the question we have that arises from this is that if you look at the fluorescence data, you see both FAM and VIC fluorescence, but the machine still chooses homozygous. Would you have any ideas as to why this is the case? I know off-targeting can occur, causing some fluorescence, but I didn't know if the extent at which we are seeing it would reflect that.

For informational purposes:

We ran the TaqMan SNP Assays (429358 [Codon 130] and 7412 [Codon 176]) designed to identify nucleotide changes at these positions. We ran two different annealing temperatures (60/64C), as suggested by BioRad, to try and eliminate the VIC fluorescence in the 130 assay (as it should be FAM+/VIC-). The layout in the document is as follows:

Lane A - 64C (A1/A2 - NTCs - 429358) (A3/A4 Brain A (20 ng) - 429358) (A5/A6 Brain D (20 ng) - 429358); (A7/A8 - NTCs - 7412) (A9/A10 Brain A (20 ng) - 7412) (A11/A12 Brain D (20 ng) - 7412).

Lane H - 64C (H1/H2 - NTCs - 429358) (H3/H4 Brain A (20 ng) - 429358) (H5/H6 Brain D (20 ng) - 429358); (H7/H8 - NTCs - 7412) (H9/H10 Brain A (20 ng) - 7412) (H11/H12 Brain D (20 ng) - 7412).

Again, thank you for your help. We appreciate your time.

Dear Naomi,

As I see in the graph from the attached doc, there are some samples which are not assigned. From my experience this is because of the problem I have described you in my previous message - differences in DNA content. It is not possible do determine from amplification graph, and because of the bigger difference of DNA which is engaged in PCR, some of the samples are clustered well, while other remain at the beginning of the graphic. Besides there is a trailing effect of the samples. My software assigned them as Heterozygots, but they were false ones.

I also started from 20 ng of DNA, and could not observed big differences compared to lower contents, up to 1.5 ng. This is until I dried out my DNA.

I haven't met off-target effect with ThermoFisher assays.

Still I couldnot understand what did you marked by FAM and what by VIC.

Also, without annotations on the graph it is impossible to guess which sample was the trouble.

And again - which genotypes were expected and which you received - are they in concordance? As on the pictures you had VIC homozygotes and hetrerozygotes ONLY - there are no FAM homozygotes - is it correct?

Kind regards

Hi, Levitchi, for the allelic discrimination, the samples which say "No Call" are no template controls (NTCs), so they had no DNA in them. We did two different assays in this plate. A1-A6 are Assay 1 where FAM is E2 or E3 (T) and VIC is E4 (C). A7-A12 are Assay 2 where FAM is E2 (T) and VIC is E3 or E4 (C). The samples run are known to be E3/E3, which was called by the program. It called VIC homozygous for Assay 2 and FAM homozygous for Assay 1, which is consistent with the E3/E3 genotype. I am concerned about the presence of fluorescence from the non-dominant fluore. For example, it will make a call of Allele 1 (VIC) (C) but there is still FAM RFUs present (at about half the intensity of VIC). I am wondering if anyone knows why this is happening or has had a similar experience.

Side note: H1-H6 and H7-H12 represent the same as Row A, but Row A had an annealing temperature of 64C while Row H had an annealing temperature of 60C. It did not resolve the opposite fluorescence/RFU issues. Rows B-G were blank--no tubes there.

Thanks again for your assistance.

Disclaimer: I have never worked with brain-derived DNA.

It is okay if there is some background fluorescence from the non-dominant fluore. The probes only differ by one base, so there is often some off target binding, even under optimal conditions. You just need to be able to distinguish a clear pattern difference between TT, TC, and CC.

I have attached some figures where I was using a Taqman assay to screen for a MetN1118Y variant. All of the samples were homozygous wild-type, but there is still some background signal from the off target probe. (There is also the stray sample that didn't work well, just so you can tell this is real data.) That is typical of Taqman assays. Some probe sets are better or worse than others, but you rarely get rid of the background completely.

I am a bit concerned that your peak fluorescence is so low, even for the on-target fluore. I usually get peak fluorescence readings in the 20 - 30 x 10-3 RFU range after 40 cycles. How much DNA are you adding? It is also possible, like Nathaniel was suggesting, that there is a PCR inhibitor in your samples.

Did you ever get this worked out? If not, IDT has a webinar coming up: "SNP genotyping on qPCR platforms: Troubleshooting for amplification and cluster separation" that might be helpful.

https://register.gotowebinar.com/rt/4010058933688571906?source=Homepage+banner

Hi, Angela. We did actually figure it out, and we were able to genotype all of our samples. Thank you for the recommendation of the webinar. We sincerely appreciate the help. Thanks!