RNA extraction and quality control of your Volvo

I got an e-mail the other day. It was from Agilent who said that my order of two custom microarray slides scheduled to arrive around the 5th of September were already finished and were being shipped from Germany to the analysis facility BEA at Karolinska Institutet (KI) now.

Guys! You're two weeks early! I haven't even gotten my own stuff (samples) together yet. Germans... How often does this happen? By experience - always when you're lagging behind. That's not what I replied though, but it's what I silently told myself.

Now you might think 'hey, you only have to fill one slide, 8 samples, how hard can it be?'. If you just thought exactly that, I'll give it to you. You're right, it's only 8 samples and it would take me 2 hours to extract. However (I hate that word, and I use it all the time), out of the 12 samples that were already extracted only 2 had enough total RNA for analysis, and don't get me started on the quality control of that RNA... ops, I already did that myself... they all failed.

At this point I have nothing. Fortunately for me, we have lots of archived RNA with corresponding DNA samples which have already been processed in the qPCR. Basically, that means that I can see in which RNA sample that I should have the right organisms and enough material. However (here we go again), that doesn't guarantee good enough quality RNA.

There are several reasons why the RNA in my samples might not be of good enough quality, and when I'm talking about quality it's rather specific and doesn't entail the comparisons between e.g. longevity of brands that we're so used to in modern consumption society. No, here, I'm talking about degradation and size of the RNA fragments. Like if you removed a few parts from your Volvo, it would still be a Volvo, but it would probably not function very well, and if the parts removed were body parts, the untrained eye might not even recognise the car as a Volvo anymore.
A similar problem arise when I have low quality (degraded) RNA. The probes in my microarray are all extremely specific (untrained eyes) and when the genes (Volvos) in my samples are missing a few parts due to degradation they will not be recognised.

Image credit: Fabian Oefner

To be short and simple, RNA is the coded message that our DNA relays for our cells to, down the line, translate and produce proteins with various tasks and properties. Naturally, the message doesn't stay intact for very long since, in most cases, when the message has been delivered and translated it has outlived its usefulness and is recycled within the cell.
So, I might just have been unlucky and the RNA in my samples was naturally degraded when the research vessel arrived (too late) on site. I might do a terrible job extracting the RNA, and perhaps, mysteriously introduce RNase (an RNA degrading enzyme) to my samples. The agitation step (shaking the samples really, I mean REALLY, fast together with tiny glass beads to crack the cells open) might shear the RNA to tiny bits and pieces. The latter is the only explanation that I can actually test.

I'm assessing the RNA quality in an instrument called Bioanalyzer 2100 (Agilent), which gives me a value from 1 to 10 (10 being the best - most intact RNA). It has developed into an industry standard since it uses an algorithm to objectively calculate and compare the amount of two different ribosomal RNAs. This way us scientists know that we will get good and reliable results and we secure the possibility of reproducing the experiment (important for e.g. peer-review and validation). Everyone is happy. Me included, if I can get good quality RNA, otherwise no.