I was at a party last night telling my non-scientist friends about my week at work. For the first time since I left my post-doc 8 years ago, I spent multiple days in the lab doing experiments!! We had a few drinks at this point, and in their lapse of judgement (thinking I may not give them a descriptive answer at a party) they asked me what my experiments were about. I did not disappoint, and told them all about my experiments.
This party was a BBQ and even though I’m a vegetarian, most of my friends are huge fans of meat. So I started by asking them about steak. If you bought a steak that you weren’t going to throw on the grill right away, you might freeze it and cook it later. When you take it out of the freezer, how do you know if the steak is still of high quality? Maybe it sat on the counter overnight before being put in the freezer. Maybe the freezer lost power during a storm, the steak defrosted and when the power came back on, the steak froze again. Maybe the steak wasn’t even good when it got to your house – maybe it sat on the butcher’s counter for a few days, forgotten about, before being put into the display case for you to buy. It may be nearly impossible to tell by just looking at your frozen steak if any of these events happened and whether or not these events would affect the steak’s quality.
This is something that we deal with daily as biobankers. To remind you, we collect human tumor samples for researchers to use to better understand diseases and to develop improved treatments. Ideally these tissue samples are very quickly frozen (as described in this blog post) and kept frozen. Like the steak, there are a lot of “variables” that may affect the quality of the tumor tissue sample – including the same issues that a steak may have – like sitting at room temperature for too long or freezing and then thawing and then freezing again. And also similar to the steak, it’s nearly impossible to tell by just looking at the tissue sample if any of these things have happened. Except, instead of affecting whether or not your dinner tastes good, with tumor tissue samples, research results that are essential for drug or biomarker development for brain tumors are affected.
How do we handle this little problem? We have to use a proxy for quality – something that we can analyze directly that will tell us whether or not the tissue sample is of good enough quality to be used for certain research purposes. In this case, the proxy is RNA. RNA is a great molecule to look at because it’s found in every cell, but it’s also unstable because of its natural enemy RNases. When these RNases are active (when the tissue sample is warm or at room temperature) they will function like little PacMans to chomp on the RNA, turning it into smaller and smaller pieces. When frozen, the RNases are inactive and can’t chomp on RNA. So if you look at how chomped up/small the RNA is in tissue samples, you can figure out whether the quality of the sample is good for your experiments.
A tiny filter (seen as the white line inside the pink tube) binds to the RNA.
This is what I did last week in the lab. How did I do this? First, I had to get the RNA out of the tissue sample. To do this, you have to separate the cells from one another, which essentially like grinding up a steak in a meat grinder. You then have to bust open the cells to get to the RNA. You do that by adding something that works a lot like soap that opens up the outer coating of the cells. From there, you can isolate the RNA by adding ethanol (an alcohol) that makes the RNA no longer dissolved in the liquid (what we call “precipitating” the RNA). From there we isolate the RNA on a column – exactly like the pink one you see on the right. The white line inside the pink tube is like an RNA filter that traps RNA while letting all the other cell bits flow through. Then you use water to get the RNA out of the filter. How much RNA do you get at the end? Imagine an eye drop worth of liquid that contains 10 micrograms of RNA. That’s 0.0001 grams and for comparison, a grain of rice weighs 0.015 grams. It’s not a lot, but it’s enough to know if your sample is good or not.
These ScreenTapes can analyze 16 RNA samples at one time. They are about the size of a long box of a glass slide or a ling box of matches
You still can’t “see” the RNA to know if it is all in one piece or if it’s been chomped up by RNases just by looking at this tiny bit of liquid in a tube. You still have to analyze it, and to do this, I used a cute little machine called a TapeStation (I honestly have no idea why it’s called this, since the “ScreenTapes” that you use for the analysis – in the photo at left – look nothing like Tapes to me). This machine separates the RNA based on size (you can see that by the black lines in the image below). There are two main sizes it looks at – these separate into to peaks (called 16S and 28S). If these RNAs have been chomped up, there won’t just be two peaks (or two black lines), but lots of little peaks of smaller sizes. This will indicate that the RNases were activated for some reason and the quality isn’t as good. In my experiments, the results were awesome and the RNA was of good enough quality for most experiments. It also meant that the tumor tissue was handled really well when it was collected and didn’t sit on the counter or get thawed and frozen again, for example.
The two peaks are a graphical image of the separation of the RNA shown by the dark lines on the right.
Now you may be wondering, “what about my steak?” I honestly cannot encourage you to do this level of analysis to see if your steak’s RNA is high quality. Then again, you just want to eat the steak – not do thousands of dollars of important experiments with it. So let’s just consider the steak a useful scientific analogy and go start your grill – I’ve heard there are some steaks in your freezer.
Thanks to Kelli and Alia for asking me what experiments I’ve been doing and inspiring this blog post.