Why are CT values in the Realtime PCR hidden after the 35th cycle ?
Why are CT values in the Real-time PCR hidden after the 35th cycle ?
Where I noticed that the CT value had been positive and then, when the last five cycles approached, it disappeared so that it showed the value is unknown
I am using Applied Biosystems 7500 Real-Time PCR System for MTB kit , and there are two target the first one JOE is Intrnal Control (IC) and The second FAM which is the positive value C+ .
But I noticed the disappearance of the FAM value, which was CT 19 and 22 for some samples, and then it became indeterminate (unknown)
While the values of the JOE have never changed as shown in pictures
Please,if anyone know the reason for this situation
with thanks and respect
Mohammad Alzeyadi
Because the value after 30th cycle is usually considered invalid. Thats why CT values above 30 are usually all taken as nil or bogus, as contamination or as no DNA amplification. I think there isnt anything wrong with your instrument. See even in your data the values are all presented until the 29th cycle.
I would suggest you to increase your DNA concentration and run the PCR again. Your positive control values should give triplicate readings around the 10-15th cycle and so should test samples. negative controls should still give a value at 29 and above.
It would be also useful to check the melting curves (or dissociation, depending on what the software calls it). It's a good way to see whether the signal that goes NA is worth investigating or not.
I my opinion, this depends on/is wassociated with your intrumental and/or software settings and have nothing to do with a potential "ivalidity of values after the 35th cycle". I would propose to check the settings for measurements and read out depiction/presentation (software settings).
Zaeema Khan
Thanks for the interesting and quick answer
In fact, I thought that increasing the concentration of DNA might adversely affect the results.
I will try to change the conditions again
I repeat my thanks for you.
@Lubomir Bednar
thank you for your interesting , I have examined the malting curves ... but I think the problem is with the software
Andreas Eisenreich
thank you for your answer
It may need some calibration, but I will try to increase the concentration of DNA and then go to check the software
thank you
I've been working with the ABI 7500 software for quite some time now and never encountered this problem. I suggest you check your settings under "Run method" before running your PCR. I included an image where you can see how to change the amount of cycles you let the machine run. I hope this helps you with your problem. Feel free to contact me if your problem persists or if you have any other troubles with the ABI 7500 software or machine.
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