Wait, what are you doing?

So, what am I actually doing for my research?

Well, in the northeastern US bats have been dying off at alarming rates due to White Nose Syndrome. Right now, researchers are speculating that the bat deaths are caused by G. destructans, a newly discovered, cold-loving, white fungus that grows on the noses and wings of the bats while they're hibernating in caves. Somehow, this is linked to the bats waking up more often, searching for food, and eventually dying of starvation (more or less...). I was curious if there was anything that could be gained by looking at what RNA 'pathways' were turned on when the bats are exposed to the fungus.

To look at what RNA pathways are expressed in a cell when it's exposed to something, like a fungus, you need to be able to compare the normal pathways to those after the cell has been exposed. (This is all a rather complicated process, but basically, RNA is the precursor to protein, so by looking at RNA pathways you can begin to see how a cell is responding to something- yes, I know I'm leaving out lots of details and really simplifying it... sorry.) For a few different reasons, I decided to use mouse cells instead of bat cells (mostly because gene chips for bats don't exist, their genome hasn't been sequenced yet). I bought a cultured cell line from ATCC, and started growing up the cells last summer (for those of you who don't know, regular cells, say, skin cells from your hand, would die after dividing only a few times. Cultured cells are essentially given cancer, so they continue to divide for more like 20 times- again with the simplifying).

Now, I have my cells that I'm going to expose, but what about the fungus? Well, that I tried to get a sample of by hiking up to the Mt. Aeolis bat cave and collecting some dead bats from outside the gate. I took these back to the lab, swabbed them, rubbed the swabbed on agar plates, put the bats in the deep freeze and tried to grow some fungus. And this is where my life in the lab goes downhill.

I grew lots of fungus over the summer. The first one seemed like maybe it was G. destructans, but then something else grew that looked more like it, so I switched over to culturing that one. I had already decided to run PCR on the samples to make sure I had the right fungus, but the PCR wasn't really working and I wasn't getting any results (ugg. Sick of explaining molecular biology terms. I'm sure Wikipedia has an article on what PCR is. Basically, it's used to amplify a strand of DNA so that it can be sequenced and compared to an existing sequence of DNA, thus verifying that you do or do not have the species you think you have). After many months of trying to fix the PCR and many months of growing the fungus, I read an article that said something along the lines of "G. destructans DOES NOT grow at room temperature." And included a picture of what it should look like. Oops. Not growing the right fungus.

Now, briefly, back to the mouse cells. They were growing and because I wanted to get things moving along, I decided to expose them to the fungus without being totally positive that the fungus was G. destructans. I did this, and afterwards, went on to try and extract their RNA. The only problem was that I extracted the RNA lots and lots and lots of times, and every time the concentration came out to about a tenth of the concentration that I needed. After many months of trying, I finally got the concentration up to about half of what I needed, but that was about the same time I realized I had the wrong fungus.

So... where does that leave me now? Well, back in December I took the bats out of the deep freeze, cut some of their wings off and put them on agar plates, which I have been trying to keep at around 14 degrees Celsius in hopes that they will grow fungus. Turns out, 14 degrees C is both the temperature at which G. destructans grows best and the temperature about half way up the stairs to my apartment (varying a bit based on the outside temperature) and so that is where they have been sitting for the past month (except when I went home to my parent's house for a few days and they lived in their bedroom, also around 14 degrees C). But, today, they moved. I took them into the lab, looked them over, and realized they're still not growing what I want them to. So, more bat wings were cut off, different types of agar were used, and now the wings are back on my steps.

Wow. That was a longer post then I had intended. Sorry everyone.
I realize I've still left a lot out, like, what happens after I get this RNA? Will I ever get this RNA? Is this a wild goose chase? When exactly will I give up and realize that many Plans at Marlboro involving complicated lab work have failed and that mine is one of them? Yeah, I'll write about some of that later. Now, bed.