Taqman QPCR help - amplification of FAM right from the 0 cycle (May/15/2006 )
I'm using TaqMan MGB probe from Applied Biosystems for QRTPCR. I'm using 5.8ITS as house keeping gene which is FAM. HOwever, I'm getting amplification of FAM in 5.8ITS right from the cycle 0, even in the No-templete control (water). It is happening every time with different set of water too. I used this same primer and probe set with DNA and it worked well. But with RNA RT-PCR I'm getting this kind of problem. Can anybody let me know what is going on. I'm new to these kind of work. I have attached a picture file of the amplification plot.
I would contact ABI
it may be something silly like a matter of dilution...? or perhaps proper threshold setting, although that seems unlikely?
or perhaps the FAM probe is not good?
anyhow, I would not guess contamination...that usually don't show up quite that early
I think is something to do with your machine setting.
Have you tried your primer and probe with another machine or other primer and probe with this ABi machine?
I'm trying to find ABI machine to test it. Hopefully I get it and test whether it is something to do with machine or probe or other.
However, if you guys have any suggestion, its appreciated.
It could have something to do with wrong background correction. I have the same problem with one of my Sybr assays. If you change from log scale to linear scale It will disappear (at least in my case). But apart from designing new Primer I have unfortunately no solution for you.
If you are using an ABI instrument (I see you are using the probe, but are you also using the instrument? Is this always assumed? Sorry that I don't know.), Montgomery might be on the right track. Have you done instrument calibrations lately? On mine, they include a Regions of Interest Calibration (to make sure the instrument isn't reading the spaces between the wells or that the CCD is not oversaturated), a Pure Dye Calibration (ensures raw spectra signals from run data can be characterized by comparing them to the raw spectra signals from pure dye standards), and a Background Calibration. You might have contamination in the heating block, which should be detectable by the background calibration.
Just some thoughts...good luck!