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I am trying to develop a method for figuring out how much of a certain mRNA is present in a bacterium. This is what I have so far:

  1. Perform a mRNA extraction
  2. Using Electrophoresis, to isolate the wanted mRNA strain (based off of size)

Now, I am not sure how I would quantify the amount of mRNA. I was thinking of using nano drop to test concentration, but I am not sure if that will give a good enough answer. What other methods could I use to quantify the amount of mRNA in my sample?

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You should be running qPCR to quantitate how much of a certain mRNA sequence is present in a sample. Remember that there are thousands to tens of thousands of different mRNA sequences present in an RNA prep, not to mention ribosomal RNAs, transfer RNAs, etc. If you then run your RNA prep out on a gel, you're just going to get a smear. Even if you cut out a portion of the gel that corresponds to the expected size of your mRNA of interest, there are going to be lots of other sequences present in that portion as well, so doing a gel purification step then measuring concentration with Nanodrop (which is quite accurate within its working range) won't tell you anything.

Quantitative real-time PCR (qPCR), on the other hand, when run with appropriate controls and standards, can give you a very good idea of approximately how many copies of your sequence of interest there are per nanogram of total RNA.

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  • $\begingroup$ So I can run qPCR with the standard curve ect... to find the number of mRNA. But how would I isolate the fragment of mRNA I want to test if a gel will not give me good results? $\endgroup$ – Adam Radek Martinez Jul 29 '16 at 14:28

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