I’m running a Real-Time PCR to amplify RNA using primers and a Cy5-labeled probe, but instead of the expected sigmoidal amplification curve, the fluorescence curve declines. What could be causing this issue, and what are the possible steps I can take to resolve it? Any suggestions would be appreciated!
Do you see amplification of your positive control?
Check the scale on the Y-axis, how "big" is the decline?
Without seeing an image, my best guess is that you are seeing the expected slight degradation over time because you are not getting any amplification of your target of interest.
What do your controls show? How about your reference/house-keeping gene?
I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?
A declining fluorescence curve in Real-Time PCR can result from probe degradation or quenching of the Cy5 signal. Proper storage and verification of probe integrity are crucial[2]. Incompatible materials in the reaction mix can also quench the signal; thus, checking reagent compatibility is advised[3]. Calibration of the PCR machine and the correct use of filter sets for Cy5 detection are essential[5]. Additionally, ensure optimal reaction conditions such as MgCl2 concentration and annealing temperature, as these can affect amplification efficiency[1]. Lastly, the presence of PCR inhibitors can alter fluorescence signals and should be evaluated[4].
Reference
[1]
Kontanis, E. J., & Reed, F. A. (2006). Evaluation of Real‐Time PCR Amplification Efficiencies to Detect PCR Inhibitors. Journal of Forensic Sciences, 51.
[2]
Tozaki, T., Ohnuma, A., Iwai, S., Kikuchi, M., Ishige, T., Kakoi, H., Hirota, K., Kusano, K., & Nagata, S. (2021). Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control.. Analytical chemistry.
[3]
Thompson, R., Duncan, G., & McCord, B. (2014). An Investigation of PCR Inhibition Using Plexor®‐Based Quantitative PCR and Short Tandem Repeat Amplification. Journal of Forensic Sciences, 59.
[4]
Funes-Huacca, M., Opel, K., Thompson, R., & McCord, B. (2011). A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis. ELECTROPHORESIS, 32.
[5]
Ghorashi, S., Noormohammadi, A., & Markham, P. (2010). Differentiation of Mycoplasma gallisepticum strains using PCR and high-resolution melting curve analysis.. Microbiology, 156 Pt 4, 1019-29 .
Dear Katie A S Burnette, this degradation happened in both positive control as well as samples.
the degradation is occuring after you have gotten your Ct value, likely your probe (i'm assuming taqman) is used up and there is less to use. I don't think you need to resolve this as it is happening so late in the cycle. run a relative standard curve with a serial dilution of the target to check efficiency, as long as your efficiency is 90-105% and you get a logarithmic signal to get a Ct then that degradation is not to worry about.
if this really concerns you then you can try decreasing the input into the reactions by 10-fold or more and see if it disappears
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