Real-Time PCR

Magazine

Do you own Applied Biosystems 7900HT? Lucky you!

Have you ever seen the Applied Biosystems 7900HT Real-Time PCR system? Without really knowing what you are looking at, you probably didn’t like it. In my opinion this is the ugliest qPCR instrument ever, grey, huge, heavy and noisy. These days also old, retired and slowly disappearing from qPCR labs worldwide. In fact, it is also the best, and I am counting in all new fancy and colorful qPCR machines with touchscreens and other attractive but unnecessary features. If you still possess it, my advice is to keep it as long as you can. Why do I think so?

The accuracy of your Cqs depend on the accuracy of measuring the fluorescence. When combining several dyes together, for example FAM, JOE, and TAMRA (or in fact any other dye combination) you never avoid a spectral overlap:

 

 

This is the trickiest task of instrument algorithms – to discriminate dyes properly. In plain language, how do you measure FAM only in your duplex qPCR if its signal overlaps with JOE? Most qPCR instrument vendors use emission filters to „cut out“ only the middle part of FAM spectrum thereby lowering the contribution of JOE to the FAM signal measured. But this is still far away from being accurate and moreover you loose sensitivity. But since this is the simplest way, these days nearly all qPCR machines use this approach.

And here we are back to good old ABI 7900HT. There are no filters at all! The CCD acts as spectrograph with continuous detection from 500 to 650 nm. No cutting-out, no lower sensitivity, you measure full spectra of all dyes from left to right. It is the only qPCR instrument ever working this way. Apparently the overlap problem is not solved by using spectrograph, it is in fact strengthened but can be and is solved later on during data analysis by employing a procedure called pure dye calibration which is a regular maintenance procedure users of ABI 7900HT are supposed to perform regularly (typically twice a year, alternatively we can perform it as a part of regular maintenance). BTW, if you wonder why Applied Biosystems has not used spectrograph in ViiA7 or QuantStudio, think of money.

Taken together, ABI 7900HT is very sensitive and most accurate and what else matters if not accuracy and sensitivity in your qPCR experiments? Should you decide to get rid of it, give me a call, we are always interested!

 

Richard Nádvorník, richard.nadvornik@seqme.eu

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