by Sarah Wiseman
RNA extractions were my main task for the week. This involved a lot of sample grinding and centrifuging, all to collect small amounts of messenger RNA. The different messenger RNAs (or mRNAs for short) present in a cell can tell us which sections of the genome are being transcribed within it. This allows us to compare the activity of particular genes between the leaves and the roots, for example. We can also use the mRNA to search for the activity of a particular gene that we’re interested in. This is done by carrying out reverse transcription (a technique which uses RNA as a template to make DNA); we can make then sequence strands of DNA to see if different plants have copies of the same genes. My extractions were the first stage in this long process.
To stop the fragile mRNA degrading, most of the extraction process had to be done on ice or (more excitingly) liquid nitrogen! Though this does have its draw backs – wheat grains which have been frozen solid are rather difficult to grind to a powder. I spent several mornings grappling with persistently intact samples and trying to thwart their escape attempts (and not always successfully – many a grain evaded my grasp, making a bid for freedom which could only result in their binning). I even managed to destroy a motor while trying to grind down the stubborn grains!
Monday and Tuesday were spent optimising the extraction protocol, an important yet dull and quite frustrating process. For someone who didn’t understand exactly what was going on it felt a lot like alchemy – making small adjustments here, using slightly different amounts of something else there for no obvious reason. As it is there are quite a few papers in which various scientists describe their methods for RNA extraction, though deciding which of them was most suitable for our extractions was a challenge. After a period of trial and error we settled on the protocol which gave us the best quality of RNA extracted so far.
The main problem with the initial extractions was the huge amounts of starch present in the wheat grains. This meant that the quantity of RNA we extracted was miniscule, even by mircobiologist’s standards! Thankfully changing the buffer solved that problem, as the before and after pictures show.
The next stage in the process is making cDNA, using the RNA just extracted as a template to create a DNA copy which can eventually be sequenced. The process itself involves more calculations and a very long wait – there is an hour’s incubation at 50oC whilst the added enzymes function.
The week ended with another induction, this one a tour of the Department’s Plant Growth Facility. The PGF is a surprisingly noisy building filled with many temperature and light controlled growth chambers, where most of the experimental plants are grown. My Arabidopsis now have their own shelf in one of the chambers and are looking rather small compared to their neighbours (which are at a much later stage of growth). Over the next few weeks I will be visiting them every few days to check on their progress, but more importantly watering them – something that it’s still best not to leave to the machines, even in 2014!
By Sarah Wiseman
‘So, what are you going to do after you graduate?’ A cruel question often posed by relatives, and sometimes even friends. It is particularly terrifying question for the soon-to-be finalist. Although a question potentially deflected by further study— ‘maybe a Masters or a PhD?’ is always going to be a better sounding answer than admitting that you don’t have a clue.
The Arabidopsis has just germinated, and is growing in one of the growth rooms beneath the Department.
Though that does raise a second, slyer question ‘Am I cut out for it?’ Undertaking your own research is a completely different kettle of fish to attending lectures and a couple of practicals a week as an undergrad. With all that in mind I arranged (in an unusually proactive move) to spend roughly seven weeks working in Dr David Hanke’s lab, within the Plant Sciences Department at Cambridge. This blog within a blog will be a record of my time there, and proof that I didn’t just disappear in the Summer of 2014…
The Game is Afoot
During this first week I have learnt many useful things; the location of the Growth Room (in the basement); how to sow hundreds of Arabidopsis seed (sterilise them in bleach then pipette evenly onto plates of agar); where the liquid nitrogen is kept (under lock and key behind the building) and more importantly, what I will be working towards over the next six weeks.
Harvesting and correctly labelling each variety – if you discover something amazing in a particular wheat sample it’s so good to anyone if you can’t remember which variety it belongs to!
All of the experiments that I’m carrying out will be investigating the role of cytokinin binding protein (CKP) in dormancy. Cytokinins are a group of plant hormones involved in cell division/growth and CKP prevents their action – halting germination or growth for a given period of time. The CKP levels in different crops play a role in the security and reliability of our food supply. Low levels of CKP reduce the length of time which potatoes can be stored without sprouting. Whilst in wheat, low levels correlate with greater pre-harvest sprouting (when the grains of wheat germinate whilst still attached to the parent plant, reducing crop yield and making useless bread). My experiments will help provide evidence required to begin conventional breeding programmes in both wheat and potatoes, in order to produce crops with a higher level of CKP.
Grand unifying purpose aside, I did very little towards those ends, mainly collecting and preparing plant material – planting the Arabidopsis, ready for harvest in four weeks or so and picking ears of wheat. It was rather a lazy week, but you have to start somewhere!