The Northern blot is the RNA counterpart of the Southern blot — a method for detecting specific RNA sequences by size-separating total or poly(A)+ RNA on a denaturing gel, transferring to a membrane, and hybridizing with a labeled probe. While qPCR and RNA-seq have largely superseded Northern blotting for routine gene expression analysis, the Northern blot provides something those methods cannot: direct visualization of transcript size, alternative splice variants, and RNA processing intermediates on a single membrane. This protocol covers the complete workflow from RNA preparation through detection.

When Northern Blotting Is the Right Choice

Northern blots are still the method of choice when you need to:

Visualize transcript size and integrity — confirms that your gene produces the expected mRNA length
Detect alternative splice variants — multiple bands indicate different transcript isoforms
Identify RNA processing intermediates — precursor and mature forms can be resolved
Validate novel transcripts discovered by RNA-seq
Assess RNA degradation qualitatively across samples
Confirm non-coding RNA expression (lncRNAs, pre-miRNAs) at specific sizes

Materials and Reagents

Working with RNA demands strict RNase-free technique throughout:

RNA samples: 5–30 µg total RNA per lane (or 1–5 µg poly(A)+ mRNA for higher sensitivity)
Agarose: Molecular biology grade
10× MOPS buffer: 200 mM MOPS (pH 7.0), 50 mM sodium acetate, 10 mM EDTA — prepare with DEPC-treated water, autoclave, store protected from light (MOPS turns yellow with light exposure; discard if dark yellow)
Formaldehyde: 37% solution, molecular biology grade (Sigma Cat# F8775)
Formamide: Deionized, molecular biology grade (Thermo Fisher Cat# AM9342)
20× SSC: 3 M NaCl, 0.3 M sodium citrate (pH 7.0)
Nylon membrane: Positively charged (Hybond-N+, Cytiva, or BrightStar-Plus, Thermo Fisher)
RNA ladder: RiboRuler High Range or Millennium RNA Markers (Thermo Fisher)
Labeled probe: ³²P-labeled or DIG-labeled DNA or RNA probe

Step 1: Denaturing Agarose Gel Preparation

RNA has extensive secondary structure that affects migration. A denaturing gel eliminates this.

Formaldehyde-Agarose Gel (1.2%, 100 mL)

Dissolve 1.2 g agarose in 72 mL DEPC-treated water by microwaving.
Cool to 60°C in a water bath.
In a fume hood, add:
- 10 mL 10× MOPS buffer
- 18 mL 37% formaldehyde (final concentration: 6.7%)
Swirl gently and pour into the casting tray in the fume hood. Insert comb.
Allow to solidify for 30–45 minutes at room temperature.

Safety note: Formaldehyde is toxic and volatile. Pour gels and run electrophoresis in a well-ventilated fume hood.

Step 2: RNA Sample Preparation

Denaturing Loading Buffer

For each sample, prepare:

| Component | Volume | |-----------|--------| | RNA (5–30 µg in ≤ 10 µL) | 10 µL | | 10× MOPS buffer | 2 µL | | Formaldehyde (37%) | 3.5 µL | | Formamide (deionized) | 10 µL |

Heat samples at 65°C for 15 minutes to denature.
Snap-cool on ice for 2 minutes.
Add 2 µL of RNA loading dye (contains bromophenol blue, xylene cyanol, ethidium bromide in 50% glycerol, DEPC-treated).
Load immediately.

Pre-Run the Gel

Submerge the gel in 1× MOPS running buffer (with formaldehyde in the buffer is optional but recommended for long runs: add formaldehyde to 0.22 M). Pre-run at 5 V/cm for 5 minutes to equilibrate.

Step 3: Electrophoresis

Load denatured RNA samples and an RNA size marker.
Run at 3–5 V/cm for 2–4 hours (until bromophenol blue has migrated 6–8 cm).
Recirculate buffer or replace periodically if not using formaldehyde in the running buffer, as the gel releases formaldehyde during the run.

Quality Check

If you included EtBr in the loading dye, you can visualize the gel under UV at this point. You should see:

28S rRNA band: ~4.7 kb (appears brighter — about 2× intensity of 18S)
18S rRNA band: ~1.9 kb
A clear ~2:1 ratio of 28S:18S indicates intact RNA

Photograph the gel with a ruler for size reference.

Step 4: Gel Treatment and Transfer

Optional: Partial Hydrolysis for Large Transcripts

If detecting transcripts > 4 kb, soak the gel in 50 mM NaOH, 10 mM NaCl for 20 minutes to partially fragment the RNA for improved transfer. Neutralize in 0.1 M Tris-HCl (pH 7.5) for 20 minutes.

Capillary Transfer

Northern blot transfer uses the same capillary setup as Southern blotting, with one key difference: use 20× SSC as the transfer buffer (not NaOH, which degrades RNA).

Soak the gel in 20× SSC for 20 minutes (equilibration).
Set up the capillary transfer stack:
- SSC reservoir with wicks
- Gel (inverted)
- Pre-wetted nylon membrane
- Pre-wetted Whatman paper (2 sheets)
- Paper towels (8–10 cm stack)
- Weight (~500 g)
Transfer overnight (16–18 hours) at room temperature.

Alternative: Downward Capillary Transfer

Faster (2–4 hours) and produces more uniform transfer. Place the gel on top of a stack of buffer-saturated Whatman paper, membrane below the gel, and dry paper towels below the membrane. Gravity and capillary action drive the buffer downward through the gel and membrane.

Step 5: Fixation

UV crosslinking: 120 mJ/cm² (Stratalinker auto-crosslink). This is preferred for nylon membranes.
Baking: 80°C for 2 hours in a vacuum oven. Preferred for nitrocellulose (rarely used now).

Mark the RNA side and the well positions with pencil. Check transfer by staining the membrane with methylene blue (0.04% in 0.5 M sodium acetate, pH 5.2) for 5 minutes, destain in DEPC-treated water. You should see rRNA bands. This also serves as a loading control.

Step 6: Probe Preparation

³²P-Labeled DNA Probes

Use random primed labeling (same method as for Southern blots — see our Southern blot protocol):

Denature 25–50 ng probe template at 95°C for 5 minutes
Label with Klenow fragment and [α-³²P]-dCTP
Purify via G-50 spin column
Denature at 95°C for 5 minutes before hybridization

Riboprobes (RNA Probes)

RNA probes (riboprobes) provide higher sensitivity than DNA probes because RNA:RNA hybrids are more stable than DNA:RNA hybrids.

Clone your probe sequence into a vector with T7 and SP6 promoters flanking the insert (e.g., pGEM-T, Promega).
Linearize the vector downstream of the insert with the appropriate restriction enzyme.
Transcribe with T7 or SP6 RNA polymerase in the presence of [α-³²P]-UTP (or DIG-11-UTP for non-radioactive detection).
Remove template DNA with DNase I.
Purify by LiCl precipitation or spin column.

Strand specificity: Use the polymerase on the side that generates the antisense strand complementary to your mRNA.

Step 7: Hybridization

Pre-Hybridization

Incubate the membrane in hybridization buffer at 42°C (formamide-based) or 68°C (aqueous) for 2–4 hours.

Formamide Hybridization Buffer (for DNA probes):

50% formamide
5× SSPE (0.9 M NaCl, 50 mM NaH₂PO₄, 5 mM EDTA, pH 7.4)
5× Denhardt's solution
0.5% SDS
200 µg/mL denatured salmon sperm DNA

Aqueous Buffer (for riboprobes):

6× SSC
5× Denhardt's solution
0.5% SDS
100 µg/mL denatured salmon sperm DNA

Hybridization

Replace pre-hybridization buffer with fresh hybridization buffer containing the denatured labeled probe. Use approximately 1–2 × 10⁶ cpm/mL for radioactive probes, or 100 ng/mL for DIG-labeled probes.

Hybridize overnight (12–18 hours) at the appropriate temperature:

DNA probes: 42°C in formamide buffer, or 65°C in aqueous buffer
Riboprobes: 68°C in aqueous buffer (higher Tm of RNA:RNA hybrids)

Step 8: Stringency Washes

For DNA probes:

2× SSC, 0.1% SDS — room temperature, 2 × 20 minutes
0.2× SSC, 0.1% SDS — 42°C, 2 × 20 minutes
(If needed) 0.1× SSC, 0.1% SDS — 65°C, 15 minutes

For riboprobes, include a post-hybridization RNase A treatment (10 µg/mL RNase A in 2× SSC, 37°C, 30 minutes) to digest single-stranded, non-hybridized probe. This dramatically reduces background.

Step 9: Detection

Same as Southern blot detection:

³²P probes: Autoradiography with X-ray film at −80°C (1–7 days) or phosphor screen imaging.
DIG probes: Anti-DIG-AP antibody → CDP-Star chemiluminescent substrate → film or digital imager.

Loading Controls and Normalization

Unlike qPCR, Northern blots need visible loading controls:

Methylene blue staining of rRNA bands (before hybridization) — simplest approach
Reprobing for a housekeeping gene (GAPDH, β-actin, 18S rRNA) — strip and rehybridize
Total RNA on a parallel gel stained with EtBr — provides a visual reference

To strip a membrane for reprobing: soak in 0.1% SDS at 100°C (boiling water) for 15 minutes, or in 50% formamide, 2× SSPE at 65°C for 1 hour. Confirm signal removal by re-exposing to film before reprobing.

Troubleshooting Northern Blots

No Signal

RNA degraded: Check 28S/18S ratio before blotting. RIN should be ≥ 7.
Insufficient RNA loaded: Load 20–30 µg total RNA for low-abundance transcripts. Consider using poly(A)+ selected mRNA.
Probe problems: Verify labeling by dot blot. Ensure probe targets an expressed exon, not an intron.
Transfer failed: Stain gel after transfer — significant residual RNA indicates poor transfer.

Smeared Signal (No Discrete Band)

RNA degradation: Most common cause. Repeat with fresh, high-quality RNA.
Gel ran too long or at too high voltage: Reduce voltage.
Probe hybridizes to repetitive elements: BLAST your probe sequence against the genome.

High Background

Insufficient pre-hybridization: Extend to 4 hours. Use fresh salmon sperm DNA (denature at 95°C for 5 minutes).
Probe concentration too high: Reduce probe amount.
Washes not stringent enough: Increase temperature or reduce SSC concentration.

Unexpected Band Size

Alternative splicing: May represent real biology. Confirm with RT-PCR using exon-specific primers.
Cross-hybridization: Increase wash stringency. Use a probe from a unique region of the gene.
Precursor RNA: Unspliced or partially processed transcripts run larger than mature mRNA.

How LabProtocol.co Can Help

Northern blot protocols involve numerous variables — gel formulation, transfer conditions, probe type, hybridization temperature, and wash stringency — all of which depend on your target transcript and probe design. LabProtocol.co generates complete, customized Northern blot protocols with calculated hybridization temperatures and recommended stringency conditions for your specific probe. Try it free and spend less time optimizing.

Summary

Northern blots uniquely reveal transcript size, splice variants, and processing intermediates.
Use 10–30 µg total RNA per lane; switch to poly(A)+ mRNA for rare transcripts.
Formaldehyde-agarose gels must be poured and run in a fume hood.
Riboprobes offer higher sensitivity than DNA probes due to more stable RNA:RNA hybrids.
Always include a loading control (methylene blue staining or housekeeping gene reprobing).
RNase-free technique from extraction through hybridization is non-negotiable.