I am doing gene expression analysis using rt-qPCR (iTaq™ Universal 2X SYBR® Green Supermix). I use Applied Biosystems™ QuantStudio™ 3 real-time PCR machine. I ran my control and treatment rat liver samples (cDNA templates) for PPAR-alpha gene target. I set Ct threshold as default. Results: The baseline start and end values of my samples (on the same plate at the same run) were different even though the Ct threshold (0.166) was the same for all of them. In specific, some samples had baseline start=3, baseline end=18; some had baseline start=3, baseline end=19. Is it a problem?
SYBR Green
Reaction volume: 20uL
Thermal cycling conditions:
50oC 2 min
95oC 10 min
40 cycles of:
95oC 15 sec Denature
60oC 45 sec Anneal/Extend
Hi,
The baseline and the threshold are two different parameters with different meaning in the run. The baseline range (start/end) depends on the background signal within each well and it is automatically subracted from the analysis because it contains the fluorescent signal not originated from the real target amplification. If the background florescence is added to the amplification fluorescence this will impair the quantification. The threshold, instead, is a unique value -automatically assigned for each run- "which reflects a statistically significant point above the calculated baselines". You can also manually set your threshold line.
Hi,
I think each amplification as its own background fluorescence, that's maybe why the baseline differs for each of them. You can try to set manually your baseline, but I guess the differences will remain. (Open one of the experiments given by the manufacturer such as "96-Well Comparative Ct Example", you'll notice what you described)
something else: you don't need this 2min 50°C step unless you have UNG in your mastermix
Hi,
The baseline and the threshold are two different parameters with different meaning in the run. The baseline range (start/end) depends on the background signal within each well and it is automatically subracted from the analysis because it contains the fluorescent signal not originated from the real target amplification. If the background florescence is added to the amplification fluorescence this will impair the quantification. The threshold, instead, is a unique value -automatically assigned for each run- "which reflects a statistically significant point above the calculated baselines". You can also manually set your threshold line.
Dear Nhu, I would simply highlight some details, which you may find helpful later on for your successive runs and may delete confusions regarding this issue.
Proper adjustment of threshold line is actually based on the baseline signals of your reaction and this is very important for getting a valid corresponding Ct. Usually we run the experiment by default (auto) baseline and default threshold and after the completion of run we adjust threshold manually for each target individually to correct for baseline (which actually is excessive background noise or technically the signals from unbound dye). Lower threshold settings introduce noise in our reaction and thus calculations will not reflect the true data, whereas the higher threshold settings jump near to plateau or linear phase where the data becomes less predictable.
If, in your reaction, you have excessive primers or excessive starting template or using samples extracted from FFPE, or templates with possible increased sequence variations within, so you will notice a high offset or noise mostly and this is my personal experience too. So, baseline fluctuations are not something very unusual. For such aforesaid cases, often we have to assign baseline values somewhat higher to get true data picture. Mostly, in a Sybr Green assay for the above-mentioned cases, auto baseline mode often fails and we get strange sigmoidal curves starting far away from the proper amplification starting points for other reactions in the same plate, so here switching back to manual baseline settings need to be done compulsorily after finishing your run, in order to achieve a proper well identified threshold for these awkward curvy amplifications.
Remember that the baseline end values must be 1 – 2 cycles before the earliest appeared amplification. And always use log view to check / adjust the threshold and use linear view to see the baseline.
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