Hello and welcome to my blog! My name is Caroline and I am a PhD student at the University of Sheffield. My research project focuses on Striga - a genus of parasitic plants that devastates harvests by infecting food crops. I am exploring the defence reactions that can make host plants more resistant against Striga. Due to my ongoing battles with anorexia, I haven't made as much progress as I would have liked but I am determined to finish the course.

This blog charts the ups and downs of life in the lab, plus my dreams to become a science communicator and forays into public engagement and science policy....all while trying to keep my mental and physical health intact. Along the way, I'll also be sharing new plant science stories, and profiles of some of the researchers who inspire me on this journey. So whether you have a fascination for plants, are curious about what science research involves, or just wonder what exactly I do all day, read on - I hope you find it entertaining!

Thursday 22 June 2017

Is there a kit for that? Getting busy in the lab...

If following a protocol is like cooking to a recipe, then RNA Extraction must be up there among making a croque-en-bouche or a deconstructed soufflĂ©. So why am I attempting such a thing? Well, so far my experiments suggest that a particular plant hormone may determine how resistant plants are against the parasitic weed Striga gesnerioides. Up to now, I have investigated this by testing whether mutant Arabidopsis plants that cannot make or sense this hormone are affected in their susceptibility to Striga. But this doesn’t tell me much about what is going on at the molecular level. Hence the RNA extraction…

RNA acts as an intermediary molecule between DNA and proteins. The DNA sequences of genes encode the amino acids that make up all the proteins in our body, from structural proteins like hair to enzymes that catalyse reactions. But DNA cannot leave the nucleus so it is first transcribed into RNA, which shuttles out of the nucleus to the cytoplasm where it is ‘read’ by ribosomes that assemble the amino acids together. The more active a gene is, the higher the rate it is transcribed and the more RNA molecules are produced. So one way to see what is going on is to capture all the RNA molecules in the cell at any given time – a ‘snapshot’ of gene activity in the host.

But it’s a tricky business for various reasons. First, RNA degrades very easily so the samples must be kept in liquid nitrogen or on ice at all times. Then there is the issue of cross contamination – from gloves, pipette tips, surfaces etc- which requires super vigilance to keep things sterile. And then there is the protocol itself: hundreds of steps, each with very precise centrifugation times and specific amounts of reagents. It’s certainly not for the faint hearted!
Just some of the things needed for RNA extraction: liquid nitrogen, ice box, fume cupboard and of course the QIAGEN RNeasy Mini Kit!

Not surprisingly, some entrepreneur spotted the gap in the market for an RNA extraction ‘kit’ (the same brains behind the PCR machine perhaps?). This jazzy coloured box, that looks like a Christmas present, is filled with all the equipment and reagents needed, all clearly labelled and even with special pink and purple tubes! But it still took me several hours to work my way through it, not helped by a stomach ache that nearly made me pass out a few times!

Did it work? To find out, I ran a sample of the precious RNA solution in an agarose gel. This uses an electric current to force the RNA molecules to move through a viscous medium, causing them to separate out depending on their size (with smaller molecules travelling faster). The gel contains ethidium bromide, which binds to the RNA and fluoresces inn UV light, allowing the RNA bands to be seen under UV light. After so many hours of work, here’s what I had to show for it:

My glorious gel photo - the 2 wells at the extreme left and right contain a reference solution called a Hyperladder, made up of fragments of known sizes. The four wells in the middle were loaded with my samples of RNA solutions.

Hooray! Because there are some bands present, I had some success at least! But unfortunately, the concentration isn’t high enough to take these samples to the next stage and work out which genes were the most active. So there is a bit of tinkering to do yet. But we all have to start somewhere…

On the side, I have also been experimenting with something new for me: hydroponics! My stock of Striga seeds hasn’t been germinating very well: the best I have managed is around 40%. This is probably because I have been using an artificial chemical called GR24 to trigger the seeds, but this is really suited for the related species Striga hermonthica, not S.gesnerioides. So I am going to try collecting the root exudates from cowpea, which is a natural host for S. gesnerioides, to see if this works any better. My little cowpea seedlings look quite content growing in their tubes at the moment, but what happens when they get bigger remains to be seen…
Look, no soil! Growing Cowpea the hydroponic way...
Meanwhile, the countdown for the Society for ExperimentalBiology’s 2017 Summer Conference in Gothenburg has begun in earnest! I have been invited to attend as a science writer and am already trying to arrange interviews for my big feature articles on Palaeogenomic DNA, Carnivorous Plants and incredible Animal Athletes. With a bit of luck, I may even be able to see a bit of the Swedish West Coast as well although the schedule is pretty jam packed! Stay tuned for more updates on that.
Here's wishing you a good end to the week - can't believe we have already had the longest day. Where is the time going???!

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