We are testing a new reverse transcriptase in our lab for RT-qPCR and have noticed that within 15 minute time lapses the cts have started to increase for the low cts samples and the high ct's samples are not even amplified. The decrease in half of the CP fluorescence signal is notable when the RT-PCR mix is kept prepared for 30 minutes and continues to decrease after 60 minutes. Can someone tell me what's going on?
Dear Alejandro Armas ,
The decrease in fluorescence signal observed over time could be due to the degradation of the reverse transcriptase enzyme, which is responsible for synthesizing cDNA from RNA templates during the first step of RT-qPCR. Over time, the enzyme activity may decrease or the enzyme may become denatured, which can lead to decreased efficiency of cDNA synthesis and subsequently lower fluorescence signals in the qPCR reaction.
If the low ct samples are showing an increase in ct value over time, this could be due to RNA degradation or poor quality of the RNA template, which can affect the efficiency of cDNA synthesis. The high ct samples not showing amplification may be due to inefficient cDNA synthesis or inhibition of the PCR reaction.
To troubleshoot this issue, you may want to try optimizing the reaction conditions, including the amount and quality of RNA template, the concentration of the reverse transcriptase enzyme, the length and temperature of the reverse transcription reaction, and the PCR cycling conditions. Additionally, you may want to check the integrity of your reverse transcriptase enzyme and ensure that it is stored properly to prevent denaturation.
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