mRNA is all in the news these days, since it’s a key component of Covid vaccines. I’ve written before about how RNA functions, but that’s all very abstract. What does it actually look like to work with RNA? It’s very much like cooking with miniature equipment: tiny smoothie makers, tiny coffee filters, and very fast salad spinners. Here a photographic tour of what it looks like to extract RNA from plants for my NC Space Grant project.
Step 1: Make a Plant Smoothie
My all samples start off as seedlings grown on a petri dish. When I harvest the plants, I flash freeze them in liquid nitrogen to preserve the RNA.
To extract RNA from the plants, first I need to grind up my frozen leaf samples. RNA breaks down quickly, especially if warm, so I keep my samples on ice until the grinding is complete. To slow the rate my RNA breaks down during the extraction process, I add some special “buffer” chemicals to each tube.
Each sample tube has a couple of metal beads in it. When I put the tubes on a shaking machine, the beads pulverize the plant material and mix it with the buffer. Like making a cocktail, my plant smoothies are shaken, not stirred.
Step 2: Filter the Smoothie like Coffee
Now I need to filter out my RNA from bigger chunks of plant material. I load my smoothies into tubes that act like tiny coffee filters. I will end up with discarded “grounds” of plant material on top of the filter and useful liquid under the filter. Since it would be hard to make dozens of tiny French presses, I use a quickly spinning tube- holder called a centrifuge. The centrifuge works like a washing machine on spin cycle. It flings liquid through the filter when I spin the tubes.
The clear green liquid on the bottom of the tubes now holds my RNA. However, this RNA juice still has some other plant gunk mixed in that I need to clean out. Some of the plant gunk settled down to the bottom of the tube, so I can just syphon off the clear liquid above it. Some plant gunk needs to be rinsed out.
Step 3: Salad Spin Out Everything Except the RNA
I put the clear part of my filtered plant juice into another tiny filter tube. At first, this filter is going to act more like a salad spinner than a coffee filter: the RNA will stay in the filter, the plant gunk that I don’t want gets rinsed out. I will rinse the RNA in the filter with several different chemicals, most of which smell like paint thinner, and use the centrifuge to again force them through the filter. This process is just like rinsing and spinning lettuce multiple times to get it nice and clean.
Step 4: Filter the RNA like Coffee
Now, I have nearly pure RNA stuck in my filter and I need to rinse it out. I use pure water, which dissolves the RNA and fling the water through with the centrifuge like in step 2. Now I have mostly RNA in a tube, but sometimes some DNA sneaks in, which can throw off my results.
Step 5: Tenderize any DNA
When people cook meat, sometimes they sprinkle it with meat tenderizer to make it less tough. The tenderizer contains an enzyme that breaks down proteins. Similarly, I’m going to add an enzyme to my RNA that breaks down any contaminating DNA. The enzyme works better at warm temperatures, so I float my samples in warm bath for 20-30 minutes. I try to time this step so it’s right before my lunch break.
After the DNA is all broken down, I do a couple more steps to deactivate and remove the enzyme, and then viola! I now have almost pure RNA dissolved in a tiny drop of water in each tube. At least I hope…
Step 6: The Moment of Truth
At this point, I don’t really know if my “cooking” has worked. I could have just water in that tube. I could have other contaminants. I need to use a magical machine called a nanodrop to measure how much RNA I have. The nanodrop has a special, very sensitive light sensor that can measure how much light passes through a drop of liquid. If I have a lot of RNA, the sensor will detect a particular pattern of light absorption.
I get a computer readout of the nanodrop results. If I get a big, swooping curve, my RNA extraction worked! If I get a little bump, I am sad. The machine always whirs just long enough to build up suspense while it reads each sample.
After several hours of work, I have a handful of vials with a few drops of purified RNA solution. Then the real fun can begin: measuring differences in RNA between plants grown under simulated “weightlessness” versus plants grown in normal gravity.