RNA extractions and the Bioanalyzer III: the solution

I finally figured it out. Naturally it was one of those mistakes that are considered "dumb" in hindsight, but it sure caused a ton of stress at the time. The problem was that I was not diluting my samples enough when I put them on the bioanalyzer. It's nothing to do with ethanol or chloroform or any of the other variables I spent the last couple months testing. I have been doing RNA extractions just fine the entire time, and then fucking up the quality assay.

Below you can see the differences. On the left there are two samples that I ran on October 1st. The desired concentration is between 200 and 5,000pg/ul and these were at a concentration of 92,448pg/ul and 54,308pg/ul. I diluted them down to the proper concentration and re-ran them yesterday (right column) and they turned out really great (RIN>8 is acceptably good, >9 is perfect).

So, what have I learned? The bioanalyzer is incredibly accurate in it's small range of acceptable concentrations, once you get outside of that range the quality goes way down. The nanodrop is mildly accurate, but can handle a huge range of concentrations. The first step is always to use the nanodrop to get a good idea of the starting concentration of a sample, then dilute the samples to fall within the range of high accuracy of the bioanalyzer. 

There are actually two types of chips for analyzing RNA with the Bioanalyzer: "Nano chips" and "Pico chips." The differences is the concentration they deal well with. Here's a picture:

                                           

Green = Nanodrop

Red = Bioanalyzer Pico chip (20-5000pg/ul)

Blue = Bioanalyzer Nano chip (5-500ng/ul)

It turns out that if I had been using the Nano chips, my RNA probably would have fallen perfectly within the range. That would have saved a bunch of time, heartache, and money, but on the other hand I wouldn't have learned the concentration lesson in such a lasting and exquisitely painful manner.

I now have nano chips which I will be using for my samples and I am going to run the rest of my samples to see whether I ruined them (as I had thought before) or they are actually usable (which it looks like they probably are). Most importantly for my personal self-confidence though, this means that my molecular technique is just fine, it was my understanding of the machines I was using that fell short. A deficit in knowledge is easier to correct and not such a blow to the ego.