We’ve talked a lot about theory stuff recently, like how we can use radioactive isotopes of elements to label molecules. Radioisotopes are versions of atoms that look & act the same from the outside but have inner demons battling within that lead them to decay and give off radiation we can detect. But most of my time is spent actually doing “wet work” – like sticking those labels on RNA to tracking them as they do things like travel through gels of interact with proteins. So I want to give you an idea of what it’s actually like to do some of the stuff I’ve talked about – so today’s post I’ll walk you through what I actually do (did)
Yesterday I radiolabeled a bunch of RNAs & today I ran a urea-PAGE gel to see if it worked (ran out of time yesterday). I labeled the RNA by using an enzyme (reaction speeder-upper) called T4 PNK – (PolyNucelotide Kinase isolated from the phage (bacteria-infecting virus) T4 – to add a radioactive phosphate group to the 5’ (“five prime”) end of the chain.
Basically RNA is DNA’s molecular “cousin” and, like DNA, each RNA letter (nucleotide) has a generic sugar-phosphate part (good for linking up to form chains) and a unique nitrogenous base (good for chains hugging one another through complementary base-pairing (this is the C:::G, A::U(T) thing that allows one strand to act as a template for the other). That U(T) thing? RNA has the letter U instead of T, but both basepair with A.
“GPS coordinates” of nucleotides are given based on numbering the positions of the sugar ring. And when nucleotides link normally, through polymerization (with the help of enzymes called polymerase which we looked at the other day http://bit.ly/2kGHfpR ) they’re left with the “beginning end” having phosphate(s) (PO4-) in the 5’ position (left arm) and the “end end” having a free hydroxyl (-OH) in the 3’ position (left leg).
But when we get DNA or RNA synthesized from a company, like when you order short stretches of DNA (oligonucleotides) to act as PCR primers – or you order custom RNAs to study – they don’t use polymerases – instead they use “solid state synthesis” where they basically tie down one end and pour in the next letter to be added (with places that you don’t want reacting yet hidden). More here: http://bit.ly/2We8e8W
And the really “weird” part is that, instead of adding letters 5’ to 3’, they add them 3’ to 5’ and they add versions of nucleotides where the 5’ has an -OH & the 3’ has the phosphate (opposite of “normal”). So, because of the way they do this, unless you specifically ask (and pay) them to, the 5’ end of the will be left with an hydroxyl group. For some things, this is less than ideal, but if you want to radiojlabel it, this is great – because it saves you a dephosphorylation step!
You see, if the RNA or DNA you want to label has a phosphate group there already, you have to remove it before you can add a radioactive version. So you have to first add a phosphatase (phosphate-remover). But if there’s already an -OH there, you’re free to start! But where to start?
The first step is resuspending it. It comes to you “dry” and you have to redissolve it. I dissolve mine in DEPC-treated water. DEPC is a chemical that kills stuff that can hurt RNA. more here: http://bit.ly/2XHJKWa
The tube tells you how much stuff’s in there. Usually they tell you both in terms of mass (e.g. nanograms (billionth-of-a-grams)) & # of copies of your thing – for this # of copies they usually report it as “nanomoles” – a mol is just like a “dozen” except that it means 6.02 x 10^23 instead of 12. So 1 mol of something, anything, is 6.02 x 10^23 somethings, and 1 nanomole is 6.02 x 10^14 somethings.
That molar stuff’s what I care about because what I’m really interested in is the # of -OHs that need to get phosphorylated. If I have 1 really big RNA I can have a lot of it mass-wise but barely any end-wise and the situation’s swapped for small RNAs (lots of ends!). So I start by using that nanomole quantity to dilute all my RNAs to a good molar concentration.
Usually I resuspend my RNA to 1mM (milimolar). 1M (1 molar) means 1 mol per liter, so 1mM means 1 milimole per liter and it’s the same as 1nmol/uL. 1mM is convenient for several reasons including the fact that you get it by adding 1uL per nanomole, and since the tube tells you nanomoles, not much thinking’s required.
If you don’t believe me, you can always dimensionally-analyze your way to it
1 nmol/1uL * 10^6 uL/L * 1 mmol/ 10^6 nmol = 1mmol/L = 1mM!
I did the first step the 1st time I labeled these (the bad thing about radio labeling is that it has an “expiration date” – 32P has a half-life of ~2 weeks, so after 2 weeks, the signal’s half as strong, and then only 1/4 as strong after another 2, etc. (it’s not like it happens at 2-week marks – it’s decaying constantly, but that’s just when you usually pass the 1/2-way dead mark).
Then it’s labeling time!
If I just have a couple, I’ll do them in “normal” eppendorf tubes (1.8mL), but if I have a lot, like yesterday, I’ll do it in a PCR strip. Those tubes only hold ~200uL (1/5 of a mL) but my reactions are only 50uL so it all its in – and the tubes are all connected which makes it easier to work with.
And speaking of making things easier… The reaction recipe I use is:
- 2uL 50uM RNA (since the reaction volume is 50uL, the final conc. is 2uM)
- 5uL 10X PNK buffer (70 mM Tris-HCl pH 7.6 (pH-stabilizer); 10 mM MgCl2 (PNK uses Mg2+ to help hold the negatively-charged nucleotides in place & stabilize reaction intermediates; 5mM DTT (reducing agent to prevent disulfide bond formation, etc.)
- 37uL water (to get the volume to 50uL
- 5uL hot ATP
- 1uL PNK
Since all the reactions are the same except for the RNA, I can prepare a “master mix” of all that “same stuff” – so that I only have to add 1 thing per RNA instead of 4 – so I premix the buffer, water, hot ATP, & PNK – and add 48ul of that directly to 2uL of RNA. I do this type of “master mix” thing a lot. It’s a big thumb-saver & tip-saver and it helps ensure that all the tubes are getting the same amount of everything and you don’t accidentally skip one of the components for one tube, etc.
When making master mixes, you always want to prepare for more reactions than you actually need – because every time you pipet, some gets stuck to the pipet tip, some can evaporate, etc. & you want to make sure you have enough. If all the stuff’s cheap, it’s good to make enough for a little more than 1 extra in case you mess up on 1 reaction you have enough to redo it.
But when the reagents are expensive and/or radioactive, I just make enough extra to account for some minor losses.
After adding the PNK-containing mastermix to the tubes, I incubate them at 37°C – this helps give the PNK the energy it needs to carry out the reaction.
The PNK catalyzes (helps make possible & quick) the transfer of the gamma phosphate (the “end” one of ATP’s 3 from ATP to the 5′-OH of a nucleic acid molecule. I use ([γ-32P]ATP (ATP with a radioactive gamma phosphate) – so the phosphoryl group that gets transferred is radioactive – so the RNA it’s added to becomes radioactive.
I have to add a lot more ATP than actually get used – because the Km is pretty low – basically this means that the PNK isn’t super eager to phosphorylate the RNA so you have to give it lots of “reminders” to do so – if it encounters a lot of ATP it has more chances.
After an hour I added an excess of cold ATP. This has a couple of functions. Firstly, it ensures that all the RNA gets a 5’ phosphate (even if that phosphate’s not radioactive) so they’ll all behave the same. And second, it fills the kinase molecules up with cold ATP instead of hot ATP so if some kinase ends up in your final RNA prep it’s not gonna radiolabel anything and cause confusion.
If you’re going to PAGE purify the RNA, you’re less concerned about this because that’ll remove the kinase, but the only further purification I do is run them through a little desalting column to remove all the excess ATP (hot & not)
I use these G-25 microspin columns. They’re like tiny, short & squat versions of those big gel filtration (aka size exclusion) chromatography columns I use for proteins – they’re filled with resin (little beads) with “Secret tunnels” that small stuff can enter (and thus they have to take a longer travel route) but bigger stuff can’t get into so they go around the beads & come out sooner. more here: http://bit.ly/2KxDEVF
And instead of letting everything go through like you do with protein SEC, you only spin it long enough for big stuff (like your labeled RNA) to come out. The ATP (and salts and stuff) get stuck in the column and you can dispose of them (in the radioactive waste).
Now I want to make sure it worked! So I run a urea-PAGE gel – it looks like an SDS-PAGE gel (the type we use to separate proteins by size) but it’s “agarose-gel-like” in the fact that we’re using it for nucleic acids. Agarose can’t make as meshy or as evenly-meshy a mesh as polyacrylamide so you can’t get good enough separation to tell apart slight length differences and resolve small oligonucleotides (short chains)
In SDS-PAGE, we use the detergent SDS to denature (unfold) proteins so that they separate by length not “shape.” RNA & DNA (but especially RNA because it’s extra -OH gives it more interaction opportunities – can have shape (secondary structure) too – and that can interfere with travel – so we erase the shape using urea and/or formamide. more here: http://bit.ly/2A9gEGG
The urea’s in the sample loading buffer that I mix with the RNA before loading it. How much to load? I want to load enough that I can get a good signal but not so much I saturate the detector. I like to load ~1000 counts/lane. So I use a Geiger counter to estimate counts/uL for each labeled product, then dilute those things to 1000U/uL, mix 5uL of that with 5uL of the 2X loading buffer, then load 2uL so that I get that 1000 U per lane. Which should give me a strong reading without a long wait.
Speaking of waiting, how long do I have to let the gel run for? I use a tracking dye with multiple dyes that run at different speeds to get an idea of how far different sized RNAs will have traveled so I know how long to run the gel for for optimal separation (I want my-sized RNAs to end up in the middle of the gel).
Once the run is done I transfer the gel to a piece of filter paper, wrap it in Saran wrap and stick it in a shielded cassette that blocks radiation from escaping. And I stick a phosphor capture screen on top that’ll capture any radiation that’s given off & “store it”
I will let it collect those signals and then I scan it in this Typhoon scanner.
Once it’s scanned I save the file for my notes & figurizing and “erase” the screen by placing it on a bright light.
I’m hoping to see nice crisp bands – if I see multiple bands in a lane, that could indicate that my RNA is partly degraded (one of the bad things about RNA is that it’s really sensitive and there are a lot of RNA chewers (RNAses) out in the world (which is actually a good thing because they protect us from viral RNAs, etc.).
That multiple-banding problem’s a problem with the actual RNA, but there are other problems that can occur with labeling – like molecules not getting efficiently labeled (you stick a lot of RNA in, but not all the ends get phosphorylated).
Here’s some troubleshooting tips… Your problem may be…
too much salt: PNK is inhibited by high salt – (50% inhibition by 150 mM NaCl), phosphate (50% inhibition by 7 mM phosphate) and ammonium ions (75% inhibited by 7 mM (NH4)2SO4) (according to NEB) – so if your sample’s salty you may need to desalt it before (and after)
small nucleic acid contaminants – if you have a bunch of short little fragments they may be small mass-wise but each one as an -OH which is the part the phosphatase cares about. if they’re small enough, desalting can remove them too
hidden ends – 5’-recessed or blunt ends can hide the 5’-OH sites that are there – loosen things up by heating the mixture (NEB recommends 10 min @70C, chill on ice, then add enzyme & raise temp to 37)
phosphatase erasing what is added – if you used a phosphatase to remove cold 5’ phosphates before you added the hot ones you need to inactivate the phosphatase so it doesn’t remove the hot ones too – some phosphatase (e.g. Antarctic Phosphatase & SAP) can be heat inactivated while others require other methods (e.g. use phenol-chloroform extraction to remove CIP or BAP)
more on radio labeling: http://bit.ly/2m5sGME
more on PCR: http://bit.ly/2KC9DDJ
more on dimensional analysis: http://bit.ly/2LR4CZA
more on nucleic acids: http://bit.ly/2FqasfN
more on molarity: http://bit.ly/2KQLw4k