What does it means to have an elevated threshold like in the attached pic? The qpcr results for this gene gives me this curves. Other genes showed the normal curves. What is this means and does it affect the results? and if so, how to solve it??
You had a problem with your experiment, all your Cts are way high, above 30.
1. Did you check your RNA quality? (purity and integrity)
2. Did you check your RT efficiency?
3. Did you check your primer efficiency?
4. Did use a very dilluted cDNA?
5. What is the Ct value for your endogenous control?
Just to add to Andre's useful pointers. Make sure your mastermix is properly mixed and kept cool and thst your template is well mixed in with the MM and that if using a plate that it has been spun down to remove droplets and bubbles.
Try to view the results with the y-axis in log scale - and re-evaluate the look of everything after doing that. Your Cq spread is from about 27.5 to 40 here - showing differential expression of this target in whatever samples you have run. The threshold in your picture was machine-generated I assume, and seems to be a good threshold for this target. When viewing the same data with the y-axis in log scale, one can more easily see that the threshold is placed correctly. If you feel the threshold line can be put at a better place, you can do this [manually] more easily with the y-axis in log scale. Generally the threshold line (which is usually drawn at ~10 stdev above background), can be manually placed within the lower 3rd of the most linear portion of the exponential region of the amplification curves (but only when the y-axis is viewed in log scale).
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