I am running qPCR on eDNA samples from rivers and have found that the vast majority of my samples show a decrease in fluorescence in my passive reference dye (ROX) while my target dye (FAM) increases (see Multicomponent Plot) . Additionally, amplification of these samples begins early on at around 16-20 cycles while my standard curve begins at 22 cycles. I am using a Taqman Internal Positive Control (VIC) as well. Overall, my plots look really strange and my standard curve has poor efficiency. What could be causing these weird results with ROX? This is my first time running qPCR.
Your curves indeed look weird. Did you check the plate for evaporation or condensation when you removed it? These problems may lead to weird and unusable curves. To avoid evaporation, the film should be glued well and its edge should not be too close to the wells. To avoid condensation, heated lid should be used; you also need to centrifuge your plate before putting it in the machine, to remove bubbles.
It seems to me that adjusting baseline cycles manually may help here. If this does not help, these results are unusable, and before repeating the experiment you may need to calibrate your PCR mashine and/or check if the lid heating is broken.
After the baseline is fixed (so you don't see the left side of your curves "diving" below zero), you should set the threshold in such a way that in log mode all curves look parallel when they cross the threshold. Your current threshold level looks way too low, so there is no actual amplification starting at 16th-20th cycle as you said. Most samples look negative, and a couple of positive ones have Ct value about 30 (if you set the threshold where it usually is in such graphs). Also, your standard curve here only has samples and no standards, so you can't use it for assessing primer efficiency.
I highly recommend you to consult your PI or a more experienced colleague in person.
Sarah Tomke Are you sure about the probe? In the sense that they amplify the correct PCR-product?
Did you try different annealing temperature?
Which termoprofile did you use?
Ekaterina Chesnokova I am using an unused machine that was purchased in 2019, which supposedly was calibrated by the manufacturer. I plan to use a second machine to compare results soon. I do spin the plates down prior to running the analysis and there does not seem to be any level of condensation above what is normal. On the contrary, there are standards on this plate--these were actually the only ones that amplified normally. My positive and negative controls also look great. It is only my river samples that look very strange. I've consulted with a few qPCR experts in my field and they cannot understand what is happening. They believe that it could be inhibitors so I plan to use a clean-up kit to see if that helps. Thank you for your suggestions and if you can think of any others given this new information that would be appreciated!
Sarah Tomke Is possible the ihnibitor coextrated during the DNA extraction phase.
I work on DNA from soil, and for me this is normal. Do you try to use BSA? Or a different kit of DNA extraction? Which value did you obtain during the DNA quantification (260 and 260/280)?
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