So I got these weird qPCR amplification curves today using cDNA from fibroblast cell culture. I extracted RNA using PureLink RNA mini kit and performed a DNase digestion on 3ug of RNA to remove any contaminating DNA (confirmed with PCR). After DNase inactivation, I reverse transcribed and used the undiluted cDNA in a SYBR green qPCR reaction. I had unusual curves where the fluorescence plateaued very early, so I thought maybe my cDNA was too concentrated. Shown below in the graph are curves. As you can see, the curves are bizarre. Any idea what might have caused this?
For the red curves the background correction didn't work. The cause of that problem may be the relatively steep (unspecific!) increase during the early cycles. This may confuse the software. Maybe you can fiddle around with the parameters used for the background correction (if the software allows). Otherwise you habe 3 options:
i) re-purify the samples
ii) use a different master-mix
iii) manually background-correct the curves (that requires a bit of math and programming)
Those look like Step One Plus traces.
If so, the red curves didn't correctly identify baseline. You can manually move what the computer uses as a baseline (cycles 3 or so to cycles 10 or so usually work pretty well, but you should look at the very raw data to decide when the signal actually starts to swing up)
The orange curves were probably not mixed very well. There might be a low concentration effect going on, but based on where the earlier curves are crossing a theoretical threshold, probably not.
Good luck!
I have had this issue before. Early amplification and low flat peaks.
I think your starting template concentration is to high. Either run dilution series or just take an aliquot of your sample and dilute it 5x and rerun to see if you get normal qPCR curve.
@David: no, this is not because of a too high concentration. You can see that two of the red curves have a ct around 24, what is perfectly fine. The other red curves likely contain too little template.
@Jochen, I stand corrected, I trust your expertise. However, if the cDNA is undiluted and still contains very little template then I do not think any manipulation at the qPCR stage is going to solve the issue.
@David, what you you mean with "manipulation at the qPCR stage"?
I agree that with no template you don't get valid ct values. As I see it, this will be the case for most of the red curves. But you can't measure where there is nothing to be measured. The initial steep looking signal increase up to cycle 10 and the slower linear increase up to the end of the PCR is not specific. I don't know the reasons for this but I have seen that this happens and is quite normal for many assays. The "normal" user does not recognize this because this trend is usually and automatically removed from the amplification curves. Here, this correction obviousely failed for the red curves.
Note that the vertical axis is logarithmic. The steep initial increase is about 0.5-0.9 units. The later increase you see around cycle 24 for the two red curves is about 5-10 units (more than 10 times as large! in the log axis this just looks smaller, because it happens at a higher absolute signal) - this indicates a signal increase that is specific for the accumulation of amplicon.
@Jochen, I mean that the majority of the samples present do not contain any physiologically significant amount of template and I don't know if it is worthwhile to play with the software settings
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