I’m hardly the first biochemist to make a pun – there’s a long history of technique naming fun! You might have heard of the western blot, but the 1st blotting technique it twas not – and “western” doesn’t refer to any directionality – instead it’s some good ole’ conviviality! Although the existence of only 4 cardinal directions would lead to some confusionality… But no matter what “direction” is in the name – the basic premise is the same – take a mix of biomolecules and separate (typically by size) then transfer to a membrane and probe to analyze. If the probe finds a match, a band you will see, but what kind of molecule will it be? For western blots, you’re looking for specific proteins, Southern blots look for specific DNA, and northern blots look for specific RNA.
The western blot is a way to test for the presence of specific proteins and it’s named after the Southern blot which tests for specific pieces of DNA – and the “Southern” name comes from the name of the scientist who invented the technique, Edwin Southern. The western blot isn’t the only technique to hop onto the direction name train – there’s a 3rd main type of blot called the northern blot and, as someone who studies small RNAs, I come across it in papers a lot (although I’ve yet to actually do it personally because I do in vitro (in a test tube) work where I know exactly what RNAs I put into my reactions)
The northern blot is a technique used to test for pieces of RNA that contain a specific sequence, utilizing the 1:1 base-pairing specificity of nucleic acids (DNA & RNA letters). Since A binds U (T in DNA) and C binds G, you can design probes that bind the thing you’re looking for with specificity! (e.g. if you’re looking for the sequence 5’-GATTACA-3’ you can design a probe that’s 3’-CTAATGT-5’ (though usually you use longer sequences that are less likely to occur by chance (like the reason you should use longer passwords).
This complementarity is super nice because it makes it WAY easier (and cheaper) to design probes for different sequences than it does to make probes for different proteins like those you use in western Blots – for proteins you usually use antibodies which are little proteins that recognize specific parts of other things – they can take a long time to produce and production usually involves injecting animals with the thing you want antibodies made to recognize, then letting the animal’s natural immune system select and amplify antibodies that recognize it as foreign. And then you have to purify those antibodies and make sure they’re specific for your protein (don’t cross-react with other proteins and give you false-positives), etc.
For antibodies, you often have to use a labeled secondary antibody to bind to the primary antibody (the one specific to your protein) in order to visualize it (the secondary antibodies are designed to recognize “generic” parts of the primary antibodies so you can use the same secondary antibody for multiple primary antibodies). But for RNA, you can just directly label the RNA – often through radiolabeling of the 5’ end (added a radioactive phosphate group to it)
The basic premise is:
- RNA ISOLATION – isolate RNA (often from cells under different conditions you want to compare
- GEL ELECTROPHORESIS – run it through a gel (agarose works well for big pieces, polyacrylamide (PAGE) works well for smaller ones), using the RNA’s natural negative charge to motivate it through the gel’s mesh towards a positive charge at the bottom -> this gel-swimming’s harder for longer RNAs (they get tangled up in the mesh more) so they travel slower so the gel acts as a “molecular sieve” to separate the RNAs by their length
- BLOT – transfer the RNA out of the gel & onto a membrane
- the gel was great for the stage at which the goal is to separate – but if you leave it in there it can start to diffuse – even without the positive charge to motivate it downwards, it can still wander around randomly a little – so you want to get it stuck on something it can’t wander through
- BLOCK – you only want specific binding so you pre-hybridize the membrane with “generic” DNA like salmon sperm DNA – no joke!
- PROBE – hybridization – add labeled oligonucleotide probe that has a sequence that complements the sequence you’re looking for
- VISUALIZE –
- if you’ve used a radiolabeled probe you expose the membrane to a screen – as the probe lets off radiation, it’s captured on the screen above it and when you scan the screen you can see where the radiolabeled thing was – more here
- if you’ve used a fluorescent probe, you shine the right wavelength of light on it to excite the fluorophores and get them to give off light you can detect.
A couple of details for those who are interested…
Unlike with the western blot, where you use electricity (with the field now applied perpendicular to the original travel direction) to transfer the molecules to their new home, with a Northern blot, if you’ve used an agarose gel, you can just stick something heavy on it – glass plates are often used as weight – if you put a stack of dry paper towels under the weight, capillary action will suck the liquid through the gel to get to those paper towels – and the RNA will get stuck to the membrane along the way. Or you can use a vacuum to help. Or, for polyacrylamide gels, which have tighter mesh that’s harder to escape from, electricity *is* used again (“electroblotting”)
Once it’s on the membrane, you want to make sure it stays there, so you can UV crosslink it (the UV light gives the RNA bases (especially the uracils) energy that excites them & gets them to react with amine groups on the surface of the membrane – they form strong covalent linkages that keep them stuck there. Alternatively, you can heat the membrane – this evaporates the water that’s been hiding the hydrophobic (water-avoiding) parts of the RNA, so these parts can now interact with hydrophobic parts of the membrane.
The northern blot used to be used a lot more, before the days of RT-qPCR. More about RT-qPCR here: http://bit.ly/2P1EXy9 But the basic idea is that you indirectly measure how many copies of a specific RNA is initially present by copying it into DNA form and making copies of those copies using POLYMERASE CHAIN REACTION (PCR) – and seeing how many copying cycles it takes to cross a threshold copy number (the more you start with the fewer copying cycles you’ll need).
PCR works by making lots of copies of pieces of double-stranded DNA – (if you want to use it for RNA, you first have to reverse transcribe it – make a DNA version of it using a reverse transcriptase – and then you can make a complementary strand of that to get double-stranded DNA that you can “unzip” (in the melt step you raise the temp so the strands separate and can be used as templates for making the complementary strands)
you provide short pieces of DNA called primers – 1 per strand – that serve as “Start points” for DNA Polymerase (DNA Pol) to travel along the strands, adding complementary deoxynucleotides (DNA letters) as they go – and they go until they fall off of the template they’re using which (after the first cycle) is where the other primer was.
With qPCR you usually use some sort of fluorescent dye or probe that gives a readout of how much DNA is present at the end of each cycle – if all goes according to plan, the amount of DNA doubles each cycle (so exponential growth) until you run out of resources – so the more copies you start with, the more copies you’ll have at the end of each cycle. You can define a “noise threshold” and then see how many cycles it takes to get above it.
Normally the primers we use are ~20nt. And DNA Pol builds onto that. So it’s good for copying bigger pieces. But I’m interested in studying really small pieces of RNA, like microRNA (miRNA) which are short (~20nt long) pieces of RNA that complement sequences in mRNA “targets.” miRNA bind to a protein called Argonaute (Ago) & guide Ago to the target to prevent that mRNA from being used to make protein. Much more here: http://bit.ly/2BuEcpr
These small RNAs are basically the size of a primer – so how can you make copies of it? There are special adapter tricks you can use, but, since the northern blot doesn’t require copy-making, it’s an attractive alternative. Another advantage of the northern blot over RT-qPCR is that you can detect differently-processed versions of RNA – “isomers” – so you can tell if RNAs have been modified by things like trimming or tailing.
There are benefits and drawbacks of the 2 techniques to consider:
RT-qPCR is more sensitive because you amplify the original sample – each cycle you double what was present in the previous cycle (since the newly made DNA pieces can act as templates). This amplification can be *good* because you can detect RNA that’s only present at really low copies. BUT this amplification can also be *bad” because when you’re amplifying the signal “artificially” you can get artifacts! When you do RT-PCR you usually assume that all RNAs get reverse-transcribed equally well and that all of the cDNAs made get amplified equally well and consistently – a doubling per cycle. but if this isn’t the case, it can seem like you started with more or fewer copies than you really did.
You don’t have that problem in northern blotting, because you measure the original directly. But you’re far from out of the woods! With Northern Blots, you don’t have to worry about amplification problems, but you do have to worry more about the “opposite” – degradation!
RNA is really unstable – especially compared to DNA – in part because of its chemical makeup – RNA’s extra O makes it more prone to self-destruction – and in part because of enzymes called RNases (RNA chewers) to chew up RNAs that are foreign or no longer wanted. more here: http://bit.ly/2XHJKWa
So one of the benefits of RT-PCR is that you stabilize the RNA by turning it into DNA. But with Northern Blotting you’re dealing with the actual RNA itself, so you have to make sure you don’t lose it!
Another “direction” you can take with blotting is the Southern blot – the OG of the blotting world. It’s a lot like the northern blot (or I guess it’s more correct to say the northern blot is a lot like it!), except that you’re probing for DNA not RNA. It was used a lot when scientists were trying to locate genes, identify genetic rearrangements, etc.
Edwin Southern invented the technique in the 1970s because he wanted to be able to isolate the genes responsible for making 5S ribosomal RNA (rRNA) – most enzymes (biochemical reaction speeder-uppers) are proteins, but the ribosome (protein-making machinery) is a mix of proteins & RNAs, with the RNAs doing the brunt of the work.And Southern wanted to find the genes for these RNAs to give them their due credit! (and study how they work…)
Since DNA is usually present as big ole chromosomes you start by chopping it into smaller pieces using restriction endonuclease (DNA scissors that recognize & cut at specific sequences), then run it through a gel. At the time, the traditional way to go about it would be to extract all those cut up DNA pieces out of the gel (basically cut out the bands and melt away the gel around them). And then bind each of those purified bands to a filter and probe each of the filters with complementary DNA probes.
Southern thought – the probing part’s good, but why not just transfer all the bands to a filter at once? As long as you preserved their relative locations you wouldn’t lose any information – you’d lose less DNA since you didn’t have to go about purifying it first (where you’d inevitable lose some yield at each step) – and you’d save a lot of time.
Southern gives credit for the “blotting” part of the name to Frederick Sanger – the name might sound familiar because he developed “Sanger Sequencing” techniques for sequencing DNA using chain-terminating nucleotides – these dideoxynucleotides can get added but not added onto because they lack a 3’ OH and by adding some terminators of just a single letter you can puzzle out the sequence. http://bit.ly/2koF5el Sanger used electrophoresis to separate RNA fragments on cellulose acetate strips, then transferred the fragments out of the strips and onto DEAE paper by “blotting through”
It’s actually kinda funny because a bunch of different scientists wanted to join the directional naming fun – but there are only 4 cardinal directions – so you ended up with a ton of methods being called “eastern blotting” which turns out to not be so funny because it can instead be confusing… As well as as some other “hybrid” directions. Here’s a sampling…
Most of the techniques the eastern blot name has been used to refer to are variants of the Western blot that look for post-translational modifications – “bells and whistles” added to the proteins after they’re made (translated) – things like phosphorylation (addition of negatively-charged phosphate groups), glycosylation (addition of sugar chains), or lipidation (addition of lipids (fatty things))
far-eastern blot: lipid analysis – this one’s a bit more different – instead of gel electrophoresis it uses TLC (thin layer chromatography) which basically uses capillary action to “suck” liquid through a special sheet that different components in the liquid interact with differently so they travel at different rates and thus separate as they go – kinda like when a piece of paper with a marker mark gets wet and the colors in the marker dye start to separate. After the TLC, the lipids are blotted onto a PDVF membrane
middle eastern blot: mRNA analysis – it’s basically a northern blot, except that the membrane is coated with “poly-U” so it specifically binds to polyadenylated messenger RNA (mRNA) (this is the type of RNA that serves as a go-between between the DNA instructions for a protein and the actual protein)
reverse northern blot: use RNA probes to bind to DNA – this is the basic idea behind DNA microarrays, which have wells containing cDNAs and then you test to see if the complementary mRNA is present in a sample to see if a gene’s getting expressed – each tiny well on a microarray plate is like a mini reverse northern blot
northwestern blot: detect RNA-protein interactions by using RNA probes to (potentially) bind to proteins on a membrane (so basically start like a western, then probe like a northern).
southwestern blot: search for DNA-binding proteins using a protein blot (the western part) and DNA probes (the southern part). so basically do a northwestern, but probe with DNA not RNA
In looking into the history of the technique, I found that, in an early example of a “preprint” Edwin Southern gave a sketch of his Southern blot to Michael Matthews who brought the technique to the US – to my workplace, CSHL. http://bit.ly/343sKOe
more on the western blot http://bit.ly/2Iy5b6r