I want to use the microscope to detect the expression of various proteins. All with excitation ranges from 495-650nM in Multiplexed Assays.
Thank you in advance
Hi, Ian, Taqman probes are typically used for DNA quantification. I'm not familiar with a functionality allowing you to directly quantify protein expression unless you mean first converting RNA-->cDNA and running cycles using a PCR machine.
Observing Taqman fluorescence on a microscope would not be possible as a Taqman probe contains both a quencher and a fluorophore. Only by complimentary strand annealing and polymerase extension can the fluorophore be released from the quencher generating measureable fluorescence. I don't think this can be done with a microscope. If so, you would need to measure the fluorescence after every round of PCR. A typical Taqman program is 40 cycles so this is a lot of measurement. A modified PCR machine like the ABI 7900 is ideally suited for this task.
I'm including a basic primer covering realtime PCR topics produced by ABI. This will help get you started.
Thank you Harley, l also want to duplicate assays that can detect DNA mutations using Taqman probes but l want to use a microscope for the optics. Do you think such an idea is feasible even theoretically speaking. Attached is a publication which has a similar flow through as the assay l intend to perform preferably with a Stereomicroscope. Sorry for the Rookiesque questions!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Ian, a microscope cannot be used to investigate fluorescence generated by PCR cycles. Determining the cycle threshold is a difficult matter and necessitates fluorescence measurements after every cycle.
Theoretically, it's possible. I envision a setup where a single PCR cycle is completed (maybe in a waterbath instead of a thermalcycler). Rather than measuring fluorescence with a microscope, which is tough to do with a PCR tube, you should use a spectrophotometer. You would need many replicated to average out fluorescence that may be generated with the added time of measurement. Replicates will lengthen the amount of time required to measure the fluorescence after each cycle.
I recommend abandoning this idea and finding a lab that will allow you to use their realtime PCR thermalcycler. Perhaps you can collaborate with a lab that has a realtime thermalcycler if you are unable to find one. You may also find utility in identifying an online service that can run realtime samples for you like Life Technologies (link included).
from the paper:
Amplification result analysis
The analysis of the results was performed using Sequence Detection
System software (Applied Biosystems) and was based on two parameters:
the cycle threshold (CT) in which the detection of fluorescence
starts, and the cumulative fluorescence signal (CFS) of each
probe at the end of the 40 amplification cycles. Any fluorescence
signal detected before the 40th cycle was considered as DNA ampli-
fication detection following Applied Biosystems indications.
https://www.lifetechnologies.com/us/en/home/products-and-services/services/custom-services/molecular-biology-services/taqman-real-time-services.html
Thank you Harley. You see, l have managed to detect DNA amplification using LC Green with the Microscope. I know its not easy it took me a year and a half.
My concern is specifically is it possible to get a Microscope unit with a Fluorescence filter wheel to view probes of different excitation energies, do you think it will still be possible? I have seen videos of how FRET works but l haven't yet come across a publication were it was done without using a commercial unit.
I am trying to get proper info before l call Microscope Reps from the suppliers..
Hi, Ian! I've heard, that in TATAA Biocenter they use probes to quantfy proteins, they call it "ImmunoPCR". But you need amplifyer anyway.
But overall idea in article, which you have mentioned above, is nice. Advise you to write to authors directly.
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