PCR amplification and the number of cycles

I had earlier decided that amplifying 8ul for 9 cycles would be good for my libraries. It turned out that 9 cycles was much to few and I increased the cycle number to 12 as per J~'s suggestion. I have now amplified all 40 samples at 12 cycles (still 8ul) and have just finished bioanalyzing the results. It seems that 12 has worked great for many of my samples, but has under-amplified a few (see below).

Of these samples, the first 4 are under-amplified, the middle 4 are fine, and the last 4 are perfect. None are over-amplified which is great. 

Next I need to pool all of these samples into two lanes as per my earlier plans. The pooling will be done so that there are equal molar ratios of each sample in the final mix. The issue I am having is that J~ and I had talked earlier and said that I should amplify each sample the same number of cycles (12) but at this point, 12 cycles has under-amplified some of them and not others. One option is to amplify the low samples a couple times more to bring them up to par with the rest. I am unsure of whether treating some samples differently will cause any catastrophic failures of my experiment. It seems like it will not as the expression analysis will be standardized by the relative expression of a number of housekeeping genes. I would guess that this should remove any effects of cycle number, but I'm not sure. I'll talk to J~ and report back.

Below is a plot of all the samples spread along the X-axis and the final concentration (ng/ul) that the bioanalyzer reported on the Y. The different colors represent different batches of library prep (batch size = 8) and the different shapes represent the different RNA extraction batches (batch size = 4). The horizontal line is at 5ng/ul.

It looks like there are no batch effects for the most part. The one main exception is in the red batch where the squares (males) are lower than the circles (females). Earlier, I had started with unequal amounts of RNA for that batch and I ended up re-doing the males. It seems that something went wrong with the 2nd prep of those, and I need to re-prep them again. As long as I am at it I will also re-prep some of the others that are below 5ng/ul.