Northern blot
Check the size and abundance of a specific transcript.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1The only common method that shows you a transcript's length
A northern blot separates intact RNA by size on a denaturing gel, transfers it to a membrane, and then uses a labelled probe to light up one transcript among the tens of thousands present. What comes back is a position on a film, and a position converts into a length.
Size
Interpolated between ladder bands, so good to a few per cent: enough to tell 2.1 kb from 4.2 kb and nowhere near enough to call a 100 nucleotide difference. It is a measurement of the intact molecule, not of a fragment of it.
Isoform pattern
Every length the probe can bind appears at once. Two bands means two transcripts, and the ratio between them within a lane needs no normalisation, because both were loaded, transferred and probed together.
Integrity, visibly
Degraded RNA shows as a smear running down from the band and as a collapsed 28S/18S ratio on the stained gel. You see that the sample is broken, rather than concluding the gene is off.
Sensitivity
No amplification step, so sensitivity comes only from how much RNA you load and how hot the probe is. Micrograms per lane, days of work, and a scarce transcript may simply be out of reach.
2Why the gel must be denaturing
RNA is single-stranded, and single-stranded nucleic acid folds. Hairpins and longer-range pairing make a molecule compact, and a compact molecule threads through agarose faster than an extended one of the same length, so on a native gel a transcript runs well ahead of where its length would put it, and reads as much smaller than it is.
3Loading, and the two bands you did not probe for
Ribosomal RNA is around 80–90% of total RNA by mass, so on a stained gel of total RNA you see essentially two things: 28S at about 4.7 kb and 18S at about 1.9 kb, with your messenger RNA as a faint smear between and around them. Those two bands do double duty.
- Denaturing gel
- Agarose containing formaldehyde, or RNA pre-treated with glyoxal, so that base pairing cannot form and mobility depends on length rather than shape.
- 28S / 18S
- The two large ribosomal RNAs, about 4.7 and 1.9 kb in mammals. Their intensity reports loading and their ratio reports integrity.
- Riboprobe
- A single-stranded antisense RNA probe made by in vitro transcription. Strand-specific, heavily labelled, and able to survive a hotter wash because an RNA:RNA duplex is more stable than DNA:RNA.
- Stringency
- The combination of salt and temperature that decides how much mismatch survives washing. Low salt and high temperature are stringent; high salt and low temperature are permissive.
- Cross-hybridisation
- A probe binding a related sequence, usually another member of the same gene family. It shows as an extra band of identical size and intensity in every lane.
- Capillary transfer
- Drawing buffer through the gel into a stack of dry towels so that RNA is carried onto the membrane. Large molecules leave the gel slowly, which makes transfer size-dependent.
- Crosslinking
- Fixing the RNA to nylon with UV or by baking. Until it is done, nothing holds the RNA on the membrane through hybridisation and washing.
- Alternative polyadenylation
- Use of a different poly-A site, giving transcripts with the same coding sequence and different 3′ UTR lengths. A common reason for two bands and a classic blind spot for a single qPCR assay.
4The probe decides what you can conclude
Two separate choices: what the probe is made of, and where on the transcript it sits. Chemistry sets sensitivity.
Key terms
Practise these as flashcards →The length of the whole RNA, read by interpolating a band's position between ladder bands on a denaturing gel. Precise to a few per cent.
The relative intensity of two bands in the same lane. Needs no loading control, because both bands shared every step of the experiment.
The standard denaturing system: formaldehyde in the gel and the sample buffer, run in MOPS, which keeps RNA extended so mobility tracks length.
An alternative denaturant forming a reversible adduct on guanine. Gives sharper bands, but the adduct comes off if the running buffer drifts above pH 8.
Reversible staining of the membrane after transfer, used to record the ribosomal bands and the ladder positions and to confirm that transfer worked.
Removing the probe so the same membrane can be hybridised again, usually for a loading-control transcript. Each round costs some RNA.
How much label a probe carries per unit mass. Along with the amount of RNA loaded, it sets the detection limit of a method with no amplification step.
The flattening of film response near maximum density, which compresses every intensity ratio measured from a long exposure.
When to use it
- Confirming the size of a transcript, or that a transcript of the expected size exists at all: the standard check when a new gene or a new construct is characterised.
- Measuring a change in isoform usage, such as a switch between alternative poly-A sites or a retained intron, where an amplification assay in shared sequence reports the sum and sees nothing.
- Showing that a knockdown or knockout removed the transcript rather than merely disrupting the region a qPCR assay amplifies, and that no truncated product remains.
- Detecting and sizing small RNAs (microRNA precursors and mature forms, tRNA fragments) where a size difference of a few tens of nucleotides is the whole result.
- Diagnosing a suspicious amplification result: a band at an unexpected size explains an assay that reports a change no one can reproduce.
Compared with alternatives
Far more sensitive, quantitative over a wide range and a hundredth of the work, but it measures a short amplicon. Prefer it for how much; come here for how long, how many forms and whether it is intact.
The same transfer-and-probe logic applied to DNA. The denaturation step there exists to make the duplex available to the probe; here it exists so that migration reports length.
Run first, and on much less material. It measures the integrity of the bulk RNA by chip or gel; a northern shows integrity only of the one transcript the probe binds, but shows it in the same experiment as the result.
Reports isoforms genome-wide and quantitatively, which a northern cannot. It infers them from short reads, though, so a northern is still the cleanest confirmation that a predicted isoform exists as a molecule of that length.
The same probe chemistry used to ask where a transcript is rather than how long it is. Complementary rather than alternative.
The protein analogue, and named after this one. Size plus specific detection, with the same dependence on transfer, blocking and a loading control.