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Matty you only need to subtract darks from flats if they go for more than a few seconds.
As for ISO it is important to take your flats at the same ISO as your lights and darks. Flats don't just help to remove vignetting, dust and other optical aberations, but also help level out the quantum efficiency differential between pixels. i.e. say your chip has a QE of 40%. That is an average across the entire chip. Individual pixels will vary from that 40%. A flat helps to level out the playing field, so to speak. Now by varying your ISO you are basically changing the gain of the camera. Changing gain changes how many electrons are used for each Analogue to Digital Unit. If you do this you will affect how the flat influences the quantum efficiency differential. You will infact introduce more noise than what you are trying to remove (flats remove multiplicative "noise"which is why they are divided).
While I have used much of Jim Solomons information over time I'll have to disagree with him in this. As part of a project on testing noise, gain and other factors of different consumer grade cameras I found a noticable difference between using ISO, particularly if there was more than one ISO setting between the images.
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