I have problems getting very weak fluorescence signal from my probe-based RT-qPCR assay. The Ct is not bad (less than 30, and I see a clear although weak band on gel), but the signal intensity is low and probably therefore the curves are not nice and clean but sort of shaky. This is a standard probe-based assay with CY3-BHQ2 probe, using the Strategene MX3005P instrument, and the default fast 2-step protocol, annealing/extension at 60C.
I have had similar problems with another CY3-based probe previously with this instrument, so I'm slightly suspecting that there might be something wrong with the detection. I have set the CY3 signal amplification to the maximum in the software but it's still quite weak.
Alternatively, I guess it might be about the probe binding. I have little experience in optimizing this type of assays, so I would welcome any suggestions here. Increasing probe concentration? Lowering the annealing/extension temp? Trying a 3-step protocol perhaps?
"Shaky" curves usually point to a dying light source. As far as I know you have a tungsten halogen lamp in your instrument like it is also used in the Bio-Rad systems. When I observed bad curves in our MyIQ5 I first did a recalibration of the system using reference dyes and when it got worse after a while I changed the lamp and after that it was ok again. If you can exclude this for your instrument, then you could also check for ozone sources in your lab. Stability of Cyanine 3 is affected by ozone. There shouldn’t be much natural ozone around in Finland at this time of the year but maybe there are other sources like e.g. laser printers around. And if it has nothing to do with the instrument or ozone then try to improve the experiment like you already suggested, annealing temps, 3-step, but increasing probe concentration you should do at last. Not in every case the more is the better. HTH, Robert
"Shaky" curves usually point to a dying light source. As far as I know you have a tungsten halogen lamp in your instrument like it is also used in the Bio-Rad systems. When I observed bad curves in our MyIQ5 I first did a recalibration of the system using reference dyes and when it got worse after a while I changed the lamp and after that it was ok again. If you can exclude this for your instrument, then you could also check for ozone sources in your lab. Stability of Cyanine 3 is affected by ozone. There shouldn’t be much natural ozone around in Finland at this time of the year but maybe there are other sources like e.g. laser printers around. And if it has nothing to do with the instrument or ozone then try to improve the experiment like you already suggested, annealing temps, 3-step, but increasing probe concentration you should do at last. Not in every case the more is the better. HTH, Robert
Hi make sure that your machine is calibrated against Cy3 dye. Many machines are calibrated against mainly for FAM, TAMRA, SYBR, CY5, ROX and VIC only
CY3 and other yellow dyes are optional for calibration. Even I faced the same problem initially .
Hi Mikael,
As Cy3 is close from VIC fluorophores for Abs/Em wavelength, I do not think it is a device calibration problem. I have experience with Cy5 PCR system, and the use of this fluorophore in duplex with FAM or VIC ones gives always a far weaker signal. So I would say that it would be interesting to test your probe with another fluorophore like VIC with stronger fluorescent signal.
Basically, It has to be checked that your PCR system and primers work well, it seems to be a trivial thing, but if your PCR is not efficient enough, your probe will have also more difficulties to hybridize. So a Sybr test with your primers to check PD and specificity would be good.
You also said that your Thyb is 60°C, if a little bit too stringent, try 58°C to see whether your hyrbidization is better, always checking that efficiency and specificity is still ok.
At the end, maybe checking, as said before, the PCR device for intensity lamp and others functions asking Agilent, if it has not been done recently.
Good luck for that
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