Is it possible to use/reprogram Open qPCR as a "smart cycler"?


#1

Hello Chai community,

I have a unique protocol that I’m trying to develop and it requires a piece of equipment that doesn’t currently exist.

I routinely need to do a preparative amplification of a template with an unknown concentration and without over-amplifying. Unfortunately, given the ranges of unknown input, optimal amplification can require anywhere from 12-38 cycles. In order to determine the correct number of cycles, we currently take a few aliquots of the reaction and run them for a staggered number of cycles, and, after running a gel to determine optimal amplification, apply that to a large scale reaction with the rest of the material. I need enough material at the end to do an in vitro transcription so it’s typically a 1.5 mL reaction divided up into aliquots.

I’m trying to find a way to spike a dye into one of the aliquots, and use a qPCR to monitor fluorescence as the samples cycle. What I need is a way to program a qPCR to, rather than run for a fixed number of cycles, monitor the reference sample and use a pre-defined fluorescence value as a trigger to finish the run and go to 4 degrees forever.

So a qPCR is actually significantly more sophisticated than the type of “smart cycler” that I actually need, and while undoubtedly not an existing feature of the software, I was wondering if this was a plausible use that one could program for this device.

Thank you for any insights or suggestions you can provide.

Also, sorry to the Chai folks for also submitting this as a support ticket, but I figured the community might be a better place to have this discussion.

  • Keith

#2

Hi Keith,

That’s a great question, thanks for sharing your application!

You are correct - the software currently does not support such a feature. However, we’ve had some requests for a similar functionality, which will be implemented in a later software release. This future feature will allow users to manually abort the protocol during the run, once they’ve decided the curves have reached the desired fluorescence level. The last cycling stage will complete through extension and move onto the next stage. In your case, the next stage would be at 4 degrees C (which you would have set initially on the protocol editor).

In the meantime, I can suggest a manual work-around. On the amplification screen of the UI, the block and lid temperatures are displayed on the bottom left corner throughout the protocol. The curves will come up in real time as target is being amplified. Once the curves reach the desired fluorescence, confirm that the last cycle completes through extension by checking the block temperature. Then click “Stop Experiment”. The run will be aborted, but since the software outputs the Cq values in real time - not just upon completion of the run - the Cq value will populate and the amplification curves will display up to the cycle you aborted.

At this point, you may take out the samples and store them at 4 C or generate another protocol that holds the current set of samples at 4 C. I understand that this may not be ideal, but it’s a possible hack before the future feature release. Let me know if you need any clarification. Happy to hop on a call with you as well.

Cheers,
Lily