Tag Archives: RNA

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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A note from the lab bench

Dear internet,

It is infuriating to discover you have slipped up somewhere and ended up with next to no DNA or RNA or whatever at the end of your prep.

It is even more frustrating when you had planned really well and done everything to the letter.

But the most frustrating thing of all must be to be certain you have done it all right, have it all go wrong anyway and then discover someone else in the lab already knows why. 

I have (had? *sadface*) some RNA. I DNA-ase treated it prior to using it in qPCR. It needed cleaning up.

In the previous incarnation of this experiment I cleaned up the RNA (Qiagen RNA MinElute) then DNA-ase treated it. (Turbo DNAfree, in case you’re interested).

Except that the more I thought about it the more I worried that I was leaving residual DNase behind. So this time around I used the Turbo DNA-ase and then cleaned it up using an RNease Mini Kit. Exactly like it says you should. And all of my beautiful RNA is… well not gone. But coming out at ~50ng / uL. FIFTY. What the actual hell?! The other way around I got nearer TWO THOUSAND!!

Now obviously a MinElute elutes into 14 uL not 30, so it’s only natural to expect the prep to be half as concentrated: let’s say it was a pretty rough prep that would have had 800, then I might expect 400. But fifty?! Absolutely gutted and so unbelievably cross with myself for throwing all 14 samples at it at once.

But the most annoying part of all? Another PhD student in the lab figured this out about 2 months ago. Turbo DNA free and Qiagen RNeasy mini just aren’t compatible with one another and as far as I can see nobody has ever made a note of this.

Yours grumpily,

Baking Biologist

Molecular Biology 101: What’s an -ome when it’s at home?

Genome

Proteome

Transcriptome

Exome

Microbiome?

All of these words are becoming part of the scientific vernacular, and new ones are arising all the time. But is this proliferation of terms meaninful, or even a good thing?  Continue reading

RNA extractions

There’s something slightly disconcerting about going back to something that used to be an every day occurrence, only to realise you’re not quite sure how to do it any more. Like the first time I got in a boat, about a year after I’d stopped rowing, and found that suddenly I was skimming my blade and forgetting to feather. Or the first time I tried to roast a chicken, having been vegetarian for the past 5 years, and couldn’t remember how to get butter under its skin. 

Today I have mainly been doing RNA extractions. For the first few months of my PhD I did a lot of RNA extractions. And in the next few months I will also need to do a lot. Hundreds in fact. If I use up all of my harvested tissue… about 500 I think? Yeah, something tells me I’m cutting down my sample size.

Anyway, I haven’t extracted any RNA in near enough a year, so today has been a bit of a trial run, and it’s made me quite nervous: realising just how much I’ve forgotten. 

Some things I have remembered today:
– Safety specs. They’re quite a good idea, but not something I regularly use while setting up PCRs, or cloning. I didn’t remember just how much I liked them until the first time I splashed some TRIzol and promptly went Ohhhh HELL. No harm done, but I quickly dug those out of the cupboard!
– Isopropanol pellets like to move. Kids, here’s a tip from your aunty BB: if you’re doing an isopropanol wash, you had better have your hand on the centrifuge as it’s slowing down. Because if you leave that supernatant in there for a minute then the pellet will decide to float away. And THAT is frustrating! 
– Liquid nitrogen burns hurt. A lot.  If I were to have a burning kleptomaniac urge to rob a bank, today would be the day to do it. Even double gloving (no really, that’s not a sex joke) my fingerprints on my left hand are definitely not quite what they used to be. 
– As much of a lab ninja you may feel when you can dissolve RNA at 3000 ng / uL, there isn’t much point when you’ll have to add 5 times as much water before you can put it down a Qiagen column. But it does make you feel ninja-y.  

And on that note, I hear the centrifuge slowing down… Toodles!