legionnaire

I haven't updated since Monday. My schedule has become bizarrely inverted. For whatever reason all of my experiments need to be done on the weekends and I've been staying up really late and waking up really late. I'm not entirely sure what it means, or why. The net result is that I'm finding weekday afternoons have become my time off, while nights and weekends I'm really busy. Not necessarily a bad way, it's just unusual and somehow out of synch with the rest of the world.

Disclaimer: If you're radically offended by the use of animals in research, you should probably stop reading here.

I've hit a good spot in my research, and I'm running with it. One thing I've learned in scientific research (I'm almost positive I've mentioned this before) is that when things are good you need to maximize what you can get out of them, because they're rarely good for very long. For about six months I've been planning this experiment to semi-quantitatively meausre the mRNA levels of different "interesting" synaptic proteins in the areas of the mouse brain that I'm studying, and compare those levels between my mutant and non-mutant mice to see if they've changed. Relatively simple in principle, but technically somewhat challenging. I'm looking over most of the lifespan of the mouse, so I've had to take samples from mice ages 1 month through 12 months (in single month increments) in pairs and extract the mRNAs and purify them. This is taking a long time because I don't have all the ages of mice that I need, so I have big groups of animals who are just sitting around aging until they're the right age for me to use. So I would take the RNAs from a single pair of animals, then wait a month and do it again. So far I only have four of the twelve ages that I need, but it's enough to try and start doing some preliminary work to see if I can get technical aspects of it up to speed.

The first thing I needed to do was check my RNA to make sure that a) it's actually there and b) that it hasn't degraded. Anyone who has ever worked with RNA knows that it's a gigantic pain in the ass - in the body it's very labile, meaning it doesn't stick around for very long, and is not that stable of a molecule. In addition, there's an enzyme called RNase that digests RNA and is all around the lab all the time because it's used in a variety of application. This enzyme has the unfortunate property of being perhaps the most stable protein we know about - you can denate it in almost any conceivable fashion by heating it, putting in highly acidic or basic solutions, radically changing salt concentrations of solutions that it's in, drying it out, etc. It will pretty much always renature itself, only to destroy all of your RNA sample. So it's somewhat nerve wracking to work with RNA, you're in constant fear of contaminating your work space with RNase and ruining all of your work.

So I had been taking samples of tissue and extracting the RNA from that tissue and storing it in our -80C deep freezer. Of course, I didn't actually know if the RNA was there, since you can't really see it. I had to run it on what's called an agarose gel, stained with a molecule called ethidium bromide that lights up when excited by fluorescent light and binds nucleic acids. A standard technique in any molecular biology lab. But I was kind of afraid to do so, because I was pretty sure there either wouldn't be anything there, or it would be a big smear on the gel (meaning it had degraded.) I was incredibly pleased to see that not only was there a ton of it there, but it was the nicest RNA I had ever purified, and it was the first time I had used this method. It's highly unusual in science for anything to work the first time.

Here's a quick brekadown of how my experiment will work - using an enzyme called reverse transcriptase (purified from retroviruses, actually) I will convert the RNA into DNA, called cDNA or complementary DNA, which is much more stable. Then, I will amplify specific cDNA molecules using a technique called the Polymerase Chain Reaction, or PCR. Read about it if you like. Only this time I'll be doing it in the presence of radioactively labeled nucleotides. After the reaction is complete I'll run out the results on a gel, and cut out the band that corresponds with the size of the RNA that I'm looking for. Then I can extract the DNA from that part of the gel and put it in a machine called a scintillation counter that measures the amount of radioactivity in the sample. If all goes well, the result will be that I will be able to see the relative amounts of different RNAs within my samples and compare them between different ages and genotypes.

This is nothing new, people have been using this technique for about ten years, but I figured if anyone was interested to know a little bit about what I do in the lab that now they know. This really has nothing to do with what I had originally intended to write about, but since I'm already out of space I guess I'll have to leave that for another day.

I'll be working all weekend, but I may be going to the anti-RNC protest on Sunday. Not sure yet. Hope you all have great weekends too.