This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control (ii) by sum of all data points in a replicate and (iii) by optimal alignment of the replicates. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. I'd be happy to have answers in the form of hotkeys, macros, or entirely different solutions.Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. Is there a way to perform the measurement step for all the rectangles in the lane at once? Second, when doing the measuring step, currently I must click on each rectangle individually, then hit m, then manually select the next one, press m again, etc., like so: Is there an easy way to move all the rectangles downward the same number of pixels at the same time? In many programs, something like Shift + Click or Command + Click would allow one to select multiple rectangles, for instance. csv (I am about to restart at step 4 for lane 2). Here, I've just completed the process and exported the results to. My immediate goal for this post is really to speed these few steps up (below), unless there is an easy generalized solution. However, there are a few key steps that if those could be streamlined, the process would become very rapid. for lane 2 and repeat above steps starting from 4.Īutomating this entire process would be best, but strikes me as a bit more involved than I have time for at present for a number of reasons. Finally, export all the measurements to a. Select each rectangle, and press `m` each time. Click on the first rectangle (from Step 5.). Repeat until every band in the first lane is selected. Move rectangle to second band -> Select next lane.ħ. Go to Analysis -> Gels -> Select first lane.Ħ. Draw a first rectangle around the first band in lane 1.ĥ. Select Edit, then `Invert` the image (such that white -> black black -> white)Ĥ. My basic procedures follows NIH recommendations for use of ImageJ. However, if there are relevant features in the standalone versions, please let me know as I would be willing to switch versions. For reproducibility, I will be using the web-based version for this post. Using ImageJ for the first time, in this case to quantify Western Blot data.
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