12 replies to this topic

[nicelady8]

Hi all,

In last RT reactions i get a positive NTC (~cycle 30). The strange thing is that I ussually load two NTC samples in each reaction, and one NTC gives no signal, but the second one, which is following my samples on plata, gives a signal. My lowest samples ended on cycle 26-27.

Is this mean I have a NTC contamination? Please help me!!!

Thank you

Natali

[prabhubct]

Did you mix NTC before taking on first one. It may be due to primer dimer. Are you getting siting curve like one in positive, if yes then might be contamination

[Trof]

If you're sure it's not a dimer, the most probable cause of a single positive NTC adjacent to sample wells on plate is cross-contamination from template pipetting.
If the contamination was in the reaction premix, both NTCs would be possitive. This however means that you have improper handling of a pipette and you may have cross contaminated other sampes as well.

Make a very strict rules about the movement of the pippete above adjacent wells. Try to move between wells or completely outside when possible. Don't release the knob unless you put the tip directly above the bin. Don't put new tip boxes in the trajectory that leads to bin. Don't splash or pipette up and down furiously.

It can still be normal contamination though, from the air, or the pipette, so generaly stick to GLP.

Run the reaction again, this time very carefully. If that still happen, clean everything and run only NTCs on a plate to check your reagents are OK. Putting NTC in wells far away from your samples would help NTC cross contamination from samples, but that would only hide the primary problem with the improper pipetting so I don't recomend that.

[prabhubct]

Close cap of NTC tubes before handling template.

[Trof]

prabhubct, on 03 September 2012 - 08:41 PM, said:

Close cap of NTC tubes before handling template.

If that's a plate, it's not possible. Also it still only hides the fact, that other samples may be cross contaminated too.

[nicelady8]

Hi! Thank you for reply.

I don't think it's an improper handling of a pipette, because I have this NTC read only resently (in last experimental set), before I had no NTC read at all. And, I do put my NTC in wells far away from the samples. Moreover, I close the tubes with NTC after adding the water to the mix  even before I begin to add my samples. Maybe I have the contamination among the samples, but it not suppose to be in NTC.

[Trof]

So, are you sure it's not a dimer? Is it SYBR assay? Is the melting curve same for samples and NTC?

[nicelady8]

Yes, it is SYBR assay, and the melting curves are the same. As I know, dimer gives two picks, but my reaction has one. Maybe I need to replace my primers anyway.

[prabhubct]

Did you check running your Realtime PCR product both NTC and positive by running on agarose gel? whether it shows primer dimer. Desired product lengths? If it is giving desired product in positive I don't think you need to reinvent wheel by designing primers again.

Edited by prabhubct, 06 September 2012 - 05:16 AM.

[Akdor]

No way, if you are having signal in a NTC and this signal is giving you the same melting temperature that your amplicon of interest your NTC is contaminated. The Melting temperature of non-specific amplifications or dimer primers should be different (usually one pick) and much lower , than the melting temperature of your product. Try to know if this signal is coming from dimers/non-specific amplification or contamination in this way. If you are having non-specific amplifications you should set up your reading step at a temperature 2°C higher that the melting temperature of the non-specific product, so you only will have signal from your specific amplicon.

[Trof]

nicelady8, on 06 September 2012 - 02:55 AM, said:

Yes, it is SYBR assay, and the melting curves are the same. As I know, dimer gives two picks, but my reaction has one. Maybe I need to replace my primers anyway.

Dimers give one peak in NTC, but what's important, their Tm is usually much lower, so you can tell. (when you have dimers and the product, then there are two peaks, but very common thing is non-template dimer, which only forms in the absence of template, it usually means you can take NTCs as negative)
If you checked also the length of the product on gel it is indeed contamination.
For that, it stays true what I wrote earlier. It's unlikely you've got contaminated reagents, because all NTCs would be positive. So.. cross contamination from pipetting, or contamination of well.

[Rajesh Chaudhary]

What I personally belive is that sometimes even it is not contamination or even if it is not primer-dimer, we can still see kind of amplification in the reaction. Which infact is not an amplification. This might be because of high amount of primer being used. Sometimes when we generate primer, it might be that it will never form the primer-dimer, for example:  the primer designed by Cawthon for telomere length measurement using real-time PCR.

But, it still shows some amplification sometimes, its because of unused primer getting jumbled which migth somehow retain the SYBR green showing some kind of amplication, which is not real.

So, if you are sure about your techniques and every steps you have gone through, there is nothing to worry about the amplificaiton seen in the later cycles. Just like you mentioned above 28-cycles. Which is fairly acceptable.

Good luck with your research!

Have a great time ahead.

[Trof]

The option of the SYBR signal originating from unused primer is interesting, however we do see band on a gel in some of our NTCs that have a signal, but different melting profile. Also SYBR only binds double stranded DNA, so primers would need to be align somehow or form a secondary structures. From this is however only a small step to a dimer. Maybe all sorts of thing happends there.

But important is it only happens in the absence of template, and that it has a different melitng profile. I would certainly worry about amplification seen in later cycles if it had the same melting profile as a product, because 28 is not unusual value for some samples. It would not be acceptable, so it's important to add melting step after each amplification with SYBR, otherwise you don't know what exactly are you looking at in the amplification graph.