High Ct value in Real Time RT PCR for NTC - Real Time RT PCR troubleshooting (Apr/14/2011 )
Hi,
I work for detection of respiratory RNA viruses by Real Time RT PCR. In some times I have experienced high CT value for NTC (No template controls) in a particular master mix. The curve is not optimal exponential type. There were more than one NTCs but others were negative. None of the samples were positive expect the little curve for NTC. My experience said this sort of false positive result may appear in Real Time PCR. But I need reference to support my claim.
My queries are,
1. Instead of considering contamination what can cause the appearance of false positive curve.
2. If a sample shows Ct value of 37-39 first time and when reextracted an repcred it turns out negative. What could be the explanation for this?
muntasir on Thu Apr 14 16:03:20 2011 said:
Hi,
I work for detection of respiratory RNA viruses by Real Time RT PCR. In some times I have experienced high CT value for NTC (No template controls) in a particular master mix. The curve is not optimal exponential type. There were more than one NTCs but others were negative. None of the samples were positive expect the little curve for NTC. My experience said this sort of false positive result may appear in Real Time PCR. But I need reference to support my claim.
My queries are,
1. Instead of considering contamination what can cause the appearance of false positive curve.
2. If a sample shows Ct value of 37-39 first time and when reextracted an repcred it turns out negative. What could be the explanation for this?
What type of assay are you running? Have you performed melting curves?
We are running real time RT PCR for detection of viruses from clinical samples. We didn't perform melting curves.
muntasir on Thu Apr 14 16:03:20 2011 said:
Hi,
I work for detection of respiratory RNA viruses by Real Time RT PCR. In some times I have experienced high CT value for NTC (No template controls) in a particular master mix. The curve is not optimal exponential type. There were more than one NTCs but others were negative. None of the samples were positive expect the little curve for NTC. My experience said this sort of false positive result may appear in Real Time PCR. But I need reference to support my claim.
My queries are,
1. Instead of considering contamination what can cause the appearance of false positive curve.
2. If a sample shows Ct value of 37-39 first time and when reextracted an repcred it turns out negative. What could be the explanation for this?
1. It depends on the probe you are using,
when you use SYBR green as its a dsDNA binding dye, it can bind to a primer dimers and can give the signal. So its essential to perform melting curve. But Taqman does not require melt curve.
Good luck
Selvam Ayarpadikkannan.
If none of your samples are giving you a positive result then I would suggest your primers are not suitable or you need to double check the concentration and integrity of your RNA prep and that you are performing the reverse transcription reaction properly, with fresh reagents.
Also double check what the requirements are for the Taq polymerase. Some require a 15 min heating step to become active which may not be part of your standard cycling program.
The signals you are seeing in your NTC sound to me like artifacts and/or primer dimers, which is why you should always perform a melting curve if you are using SYBR green.
We are using TaqMan kits for Real Time RT PCR. Our samples were negative because samples not from epidemic season. Positive controls were nice with Ct value around 30 each time. We have tested with positive known samples also. but some time this problem appear specially for the initial well of 96 well pcr block (CFX 96 by BioRad)