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
soufflé. 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!
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| 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:
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| 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…
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| 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???!