What does it mean if RT-PCR machine from thermo fisher writes threshold algorithm failed and exponential algorithm failed for some samples? How can I correct it, or should I ignore the results and not trust? How frequently should i ignore this when it happens?
Hello David,
RT-PCR threshold algorithm failed indicates there is no amplification in sample or baseline adjustment is not proper if doing manually. The probability might be issue with non suitable primers or concentration of primer is too low, annealing time or temperature is not optimal (this can be confirmed by melt curve, product amplicon size check through gel run). Another probability may be there is not sufficient initial mRNA copy in sample so that during 40 cycle of amplification is unable to detect fluorescence beyond fluorescence of reference dye. Do confirm each and every probability.
Along with your question please insert picture of actual log amplification plot and melt curve for better prediction.
Hope this help you.
Do you actually have any amplification when you get this error (or is there no amplification as suggested above)? As suggested above, an image of the amplification curve is needed to provide any advice.
Try running three different concentrations of sample to get a true baseline, the concentration of the primer is too low.
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