Help: Very odd amplification graphs (RTPCR) - (Aug/16/2010 )

Hello, I have been scouring the web for something that looks similar to these graphs that I've been getting from a RT-PCR experiment.

The template was blood serum RNA that was converted to cDNA and multiplexed with 4 primer-pairs, diluted 1:10 and run on RT-PCR with individual primers. We are thinking it may be primer interference from not diluting enough, but other than that we have no idea and have never seen this before. We did this same experiment from cell RNA and it looked quite normal. We have been having big trouble getting usable results from these blood samples.

The first picture shows amplification which crosses the threshold line and then dips back down below it...very odd.

The second picture shows two plateaus which is also odd.

Looking for any input as to what is going on. Thanks!

edit: added another picture of the dipping phenomenon.

You may have to adjust the baseline manually. To me it looks like your target is very abundant (low CT-values) and you have to lower the stop value for your baseline which is usually to cycle 15 in our cycler. Change the view on the y-axis to linear and adjust the stop value one to two cycles before the earliest amplification can be detected. Maybe that helps.

Hi

What do your curves look like if you change the scale of the Y axis? I had a similiar thing happen to me and realised my scale had changed.
Clare

Clare on Wed Aug 18 11:34:56 2010 said:

When I change it to linear like the other guy said it makes it even worse. Or do you mean something different when you say change the scale?