mRNA expression ratio

Dear Sir,

I would like to analysis the ratio of mRNA expression verus scramble mRNA expression using cell profiler.
My fluorescent image files are as uploaded.
Please HELP!

V J Amatya

Hi VJ,

You haven’t mentioned whether mRNA expression (which I assume is in the form of puncta in the cy3 image?) is to be quantified as the number of puncta per cell, or the puncta intensity (or something else?). CellProfiler can do either of these, but I would recommend starting with the speckles pipeline from our Examples page (search for “speckles” on that page) and adjust it to suit your assay.

A few comments:

  • CellProfiler can calculate a ratio of a measurement obtained from a single image to another measurement from the same image. In your case, it appears that you have one set of images for regular mRNA and another for scrambled mRNA, and so the typical CellProfiler approach will not work. You will need to calculate the mRNA expression for all your images, collect the measurements as a spreadsheet (there is a module for this), and then calculate the ratio based on the aggregate statistics using Excel or some other software.
  • I also notice that your DAPI images are saturated in areas (i.e, the camera exposure is too high), which can hamper nuclei detection. You may want to decrease the exposure to a value to allows for more uniform contrast.