Progenesis SameSpots is one of the tools that are indisputably required - providing the image and subsequent statistical analyses that are critical to a conclusive and compelling proteomics study.
How does Progenesis SpotCheck work?
Progenesis SpotCheck helps you objectively validate that your gel running meets your lab's quality standards. It also highlights some common problems that occur during image capture.
Initially, you create a Progenesis SpotCheck gold standard — a high-quality, single-sample experiment with associated stringency requirements. Progenesis SpotCheck will use your gold standard as a baseline to objectively measure the quality of your gels.
Once you have created your gold standard, you can begin testing gels that have been run using the same sample and protocol. Test gels can be those run by other staff to ensure that gel quality is maintained throughout your lab.
When testing a gel against a gold standard, SpotCheck will consider each spot individually, comparing its value on the test gel with the range of values for that spot on the gold standard gels. This is done using standard deviation to get a measure of the spread of values for the spot in the gold standard and seeing where the value for the spot on the test gel lies within that spread. (Note that the values used for this are the logged normalised spot volume measurements.)
Consider the example below which shows typical values for a single spot. The gold circles represent measurements for that spot from the gold standard, (in this example the gold standard contains 10 gels). The blue triangle represents the measurement for that spot on the test gel. The horizontal blue line shows the mean value of the gold standard measurements and the red lines show distances away from the mean at ±1 standard deviation, ±2 standard deviations, and ±3 standard deviations.
In this case we can say that the spot on the test gel is just over 2 standard deviations away from the mean of the gold standard measurements.
When you create your gold standard you can specify the criteria that will be used to decide which spots will be passed by SpotCheck, i.e., whether the above spot at 2 standard deviations from the mean is acceptable or not. When choosing these criteria you should consider the normal distribution (bell curve) of values:
This shows that, when performing tests against a spot in the gold standard we'd expect the test value to be within 2 standard deviations of the gold standard mean 95% of the time if the test gel is of the same quality as the gold standards.
To get the best results from SpotCheck you should also check the variance of spot values in your gold standard. The easiest way to do this is to open the gold standard experiment in Progenesis SameSpots and go to the CV Graph.
A good reference experiment should have most spots with a low CV and very few with higher values like the example below.
When you have completed the SpotCheck workflow to test a gel against your gold standard you will be presented with a view of the test gel with the gold standard spot outlines overlaid on top of it. You will be able to visually and numerically review the test gel and will be provided with feedback about whether or not the test gel passes the gold standard criteria.
