Real-time PCR probe degradation - when do they degrades? (Sep/12/2005 )
Hi u all.
I'm new in using probes in real time PCR and I think one of the probes I'm using id degraded. this probes is duolabelled (FAM and TAMRA) and it has not the normal sigmoidal curve on the real time plot but it goes straight after the CT ; moreover it does not give a value for the target gene similar to the one of the experiments before but it's lower. what I would like to now is: what are the most frequent causes of probes degradation? I'm using 2 probes and I kept them in the same refrigerator (-20°C), I made aliquotes of diluted probes to prevent multiple defrosting and I treated them in the same way but the second probes shows no degradation pattern... can anyone help me with some advice?
I don't think it is degradation of your probes, but rather some sort of inhibition in your pcr reaction. It sounds like you have run this before, so your primer/probe concentration are probably ok. Perhaps your DNA prep maybe not clean (contain some pcr inhibitor).
thank u for the advice, I'll try to investigate if there's any inhibitor in the reaction but i'm sure it's not the DNA because is the same I use for the reactions with the other primers/probe.
I have found that some probes will begin to degrade around 5 or 6 months. I aliquot mine and store them at -20; last year when I was a newbie at qPCR at about 5 months I noticed that one of my probes stopped working...crappy results, bad/no amplification, loss of specifity...I tore my hair out doing control after control trying to find the problem; it only happened with one of the 5 probes initially, then began to spread to the others...I was going nuts until I spoke with a very knowledgable tech support guy at NEB who said probes can degrade after awhile.
So, now I resuspend them in TE instead of H2O, and I still order up a new batch about every 6 months to preserve integrity. Primers are cheap, real-time runs are not
Have you get you FAM/TAMRA probe work before?
What real-time cycler that you use?
Are you running duplex? ( as you mention you have two probes)
Have you run a gel to confirm that your PCR product was being amplified?
If your probe do work befoe and now give you a straight line after the CT, yet your another probe give you a sigmoid curve, it indicate that you FAM/TAMRA probe has a problem.
Is either i) your probe degraded due to DNase or repeat thowing or ii) your probe is bleached due to over expose to light while you preparing you master mix. Do you store them in dark. Sometimes the probe bleaching effect also can be cause by the lausy manufacturer which I experienced before. What company synthesis your probe. I recomend Proligo.
Actually you can do a little test on your own. Expose you problematic probe under correct wave length and take a photo. you will see the better quality probe (unbleached probe) will give you higher light intensity. Biorad icycler has this function.
Or else yo can mix your probe (final concentration = 0.25uM) with 1 unit DNase 1 (togather with DNase 1 buffer) and incubate at 37oC in your real-time cycler. Your undegraded probe will give you a decrease signal after sometimes, whereas degraded probe will give you a constant signal.
By carrying out both tests, you may know the current condition of your probe.