Tag Archives: qPCR

When science gives you lemons… find out if lemonade exists

Some days, life throws you curve balls. The lights are all red; Sainsbury sell out of orange milk; the weather forecast is clear and then it heaves down. (Seriously though, this is Britain. Who am I kidding? If I leave the house without a raincoat, it’s my own stupid fault).

More frequently – in my case at least – science throws me curve balls. In fact, what am I saying? I can’t even remember the last time science threw me a straight ball.

For the last two days I have been sat sadly staring at this data. There’s a special kind of paralysis that comes upon a PhD student when an experiment might be failing and they’re just not sure: it’s the ‘I won’t know until I’ve finished it… but I don’t want to waste my precious precious samples if I could fix it instead’ sort of feeling. This isn’t really a data set yet, just two qPCR plates. The first one looked a bit funny, but the standard curve just plain didn’t run, so I was planning to discard the plate and run a new replicate. Then the second plate came out looking exactly the same. (The green line is the same samples against a different housekeeping gene).

I am looking at the expression of a gene in the wild type plant, three single deletion lines and a double deletion. (I work with a polyploid, so by crossing and then selfing the deletion lines, multiple deletions can be stacked). I expected that either the plants would show reduced expression (because they are missing a copy of the gene), or that they would be the same as the wild type: because the plant was compensating for the loss.

These are the differences in Ct values between the gene of interest and the housekeeping genes: i.e. how many more cycles did it take for the sample to fluoresce. A value of 3 is more or less equivalent to a ten times difference in expression, so really I guess I was expecting to see a difference between lines of 1 cycle (or about a third). High values mean low expression. Low values mean high expression.

Deletion line 3 is doing what I expect it to: the delta Ct value is higher, because expression of my gene is lower. So far, so good. Expression of Deletion line 2 is static. Okay, so this one is compensating. Also good. The two deletion lines are different: that’s interesting.

Uh… hold on a second. What is deletion line ONE doing? And more to the point, what is the DOUBLE deletion line doing?

*cue freak out*

How on earth can a plant that is missing a copy of a gene then be producing MORE of the transcript associated with that gene?!

For the last day I have sat and pondered. And by pondered I mean I have tried to write an application, I have marked some A-level work, and I have tried to write a chapter for FastBleep Biology that I may never finish at this rate. Today, I cracked. I told my labmates – in a slightly hysterical voice – what my data looked like.

And without batting an eyelid, one of them said “You’ve deleted a regulator.” … I’ve pardon me? “You’ve deleted a regulatory element. Gene expression is no longer being suppressed. In fact if the double deletion is doing the same thing then that sounds even more like the answer.

There’s an answer. Not only is my science not failing dramatically, but there may even be something interesting going on.

Hello science. We meet again.

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Molecular Biology 101: qPCR when you need to detect duplication events

Why are you in work so late?! asked my housemate’s boyfriend, when I explained via Facebook chat that that was where I was.

I got really into writing. And now I’ve been here so long that the qPCR I planned to run over night is done, so I may as well do the analysis! I replied

Dedication to the cause! Came the reply. But I have no idea what qPCR is…

I pride myself on talking about science far too much and boring everyone around me, especially housemates and their boyfriends. So how have I possibly avoided explaining what qPCR is?!

DNA Replication

 DNA exists in relatively small quantities compared to how much we need to do molecular biology. In order to work with it – or, for that matter, check whether it is there – we need to make more of it. A particularly awesome feature of DNA makes this possible. It is a double stranded molecule: if you could straighten out the helix that it works itself into it would look like a ladder, and the two sides are inverse copies of one another. (Once you have finished marvelling open-mouthed at my non-existent artistic skills you will, I’m sure, spot that pink always pairs with orange etc).

This means that if you split the molecule in two, it’s possible to rebuild the other side from the side that you have. This happens inside your cells all the time and is called semi-conservative replication.

The Polymerase Chain Reaction

We can simulate this in the lab through a reaction called PCR (Polymerase Chain Reaction). PCR can best be summed up by my favourite geeky advert of all time. (A geeky advert so immense that for my first 4 months as a PhD student I drank from a BioRad mug for no other reason).

In case that was all a bit much for you, here’s a quick recap:

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The mysteries of lab packaging

I’m sure I’m not alone in having regularly received lab supplies – especially enzymes – shipped in bizarrely large boxes. Today’s offering, however, really takes the biscuit:

This box (with autoclave tape for scale) contained four of these tubes (with my hand to scale). In fairness, it was shipped over the weekend, but all the same that is a shedload of dried ice for four very small tubes!

Amusingly, today I also received this.

This is also a qPCR enzyme and also says that it should be stored at -20C. This one however was shipped in a plastic bag with a (completely melted) ice block. Fingers crossed it still works… not planning on using any precious samples to test it though!

#summergoals: End of July evaluation

A few weeks ago I decided to get a handle on my summer by setting myself some goals (thanks to  Flora Poste for giving me the idea!) . I’m a very target driven person and also prone to floundering without a schedule (someone remind me why on earth I wanted to do a PhD?) The aim of the game was to make SMART goals (Specific, Measurable, Attainable, Relevant and Time-bound). In other words I set myself a whole bunch of tasks to be achieved; some big, some small; that would help my PhD in a noticeable way, and that were supposed to be complete by certain deadlines. The end of July was one of these deadlines.

My first recap of #summergoals was pretty successful. The end of July… less so.

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qPCR standard curve FAIL

Several months ago I agreed to give a talk next Tuesday. At the time it seemed a given that by then I would have some beautiful qPCR data. And then my minus 80 freezer died, taking with it all of my vital tissue samples, which took a further 6 weeks to regrow before I could even think about RNA extractions and DNase treating and cDNA synthesis and qPCR.

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