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Protocol for Small RNA Northern Blots: High-Sensitivity, High-Resolution

CLYTE research team
6 days ago
5 min read

In the age of high-throughput sequencing, the Northern blot remains the indispensable "gold standard" for validating and characterizing small non-coding RNAs. While RT-qPCR can quantify known sequences, only a Northern blot can simultaneously confirm the precise size, detect isoforms, and quantify the expression of small RNAs like microRNAs (miRNAs), short-interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs).

This article summarizes and synthesizes expert-level protocols from leading academic labs and suppliers. It provides a detailed, optimized workflow focused on achieving the high sensitivity and resolution required for detecting these <40 nucleotide transcripts.

You might also be interested in this western blot protocol! Ask Sophie to help you with Northern Blotting!

The Core Workflow: An Overview

At its core, a small RNA Northern blot follows six key stages. Success hinges on meticulous technique at every step, as RNA is highly susceptible to degradation.

RNA Isolation: Obtaining high-quality, intact total RNA is critical.
Denaturing Gel Electrophoresis: Small RNAs are separated by size using a high-percentage denaturing polyacrylamide gel (Urea-PAGE).
Transfer: The separated RNA is transferred from the gel to a solid-support membrane (e.g., positively charged nylon).
Cross-linking & Immobilization: The RNA is permanently fixed to the membrane.
Probe Hybridization: A labeled, complementary nucleic acid probe (oligo) is introduced, which binds only to the target RNA sequence.
Washing & Detection: Unbound probe is washed away, and the signal from the hybridized probe is visualized, typically via phosphorimaging (for radioactive probes) or chemiluminescence (for non-radioactive probes).

An Optimized High-Sensitivity Protocol for Small RNA

This protocol integrates key optimizations for maximizing the detection of low-abundance small RNAs.

Step 1: RNA Isolation

Begin by isolating total RNA from your cells or tissue using a standard method (e.g., Trizol reagent). For small RNA detection, 5-10 µg of total RNA per lane is a common starting point.

Step 2: High-Resolution Denaturing Gel Electrophoresis

Standard agarose gels cannot resolve small RNAs. You must use a denaturing polyacrylamide gel (Urea-PAGE).

Gel: A 15% Urea-PAGE gel (e.g., 19:1 acrylamide/bis-acrylamide) is typical.
Buffer: Use 1x TBE (Tris-Borate-EDTA) or 1x MOPS buffer.
Loading: Mix the RNA sample with an equal volume of 2x RNA loading dye (e.g., 95% formamide, EDTA, and tracking dyes like Bromophenol Blue). Heat at 70-80°C for 2-5 minutes to denature, then immediately place on ice.
Pre-running: Pre-run the gel (e.g., at 65 mA) until it reaches a stable temperature (e.g., 50°C). This ensures even migration.
Running: Run the gel at a constant current or wattage (e.g., 40 mA) until the Bromophenol Blue tracking dye (which runs at ~12 nt) is near the bottom.

Step 3: High-Efficiency Transfer & Cross-linking

This is the most critical step for small RNA retention.

Transfer: A semi-dry transfer apparatus is highly efficient for small RNAs. Transfer the RNA from the gel to a positively charged nylon membrane (e.g., Hybond-N+) in 1x TBE buffer. A typical transfer runs at ~250-300 mA for 1-2 hours.
Cross-linking (The Key Optimization): Conventional UV cross-linking (autocrosslink function in a Stratalinker) is inefficient for small RNAs (<40 nt). A chemical cross-linking step using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) has been shown to increase signal detection by up to 50-fold.
- After transfer, place the membrane RNA-side-down on Whatman paper saturated with freshly prepared EDC solution (e.g., in 1x methylimidazole buffer).
- Incubate at 60°C for 15 minutes to 2 hours.
- Rinse the membrane several times with dH2O and bake at 80°C for 10 minutes (or proceed directly to pre-hybridization).

Step 4: Probe Labeling (Radioactive vs. Non-Radioactive)

You must detect your target with a highly specific, labeled oligonucleotide probe.

Radioactive (Highest Sensitivity): Use T4 Polynucleotide Kinase (T4 PNK) to end-label a DNA oligo probe (typically ~20-22 nt) with [γ-32P]ATP. Purify the labeled probe from unincorporated label using a size-exclusion column (e.g., P-30 column).
Non-Radioactive (Safer, More Convenient): Use probes labeled with biotin or other haptens. These methods offer excellent sensitivity (sufficient for ~5 µg of total RNA) and the major advantage of long-term probe stability and avoiding radioactive waste.

Step 5: Hybridization & Washing

Pre-hybridization: Incubate the membrane in a hybridization oven with a pre-hybridization buffer (e.g., ULTRAhyb™ buffer) for at least 30 minutes at the calculated hybridization temperature (often 37-42°C for small RNA oligos).
Hybridization: Add the heat-denatured probe to fresh hybridization buffer and incubate with the membrane overnight, rotating in the hybridization oven.
Washing: Wash the membrane to remove unbound probe. This is done in steps of increasing stringency (lower salt concentration, e.g., 2x SSC -> 0.1x SSC) and temperature. Stringent washes are critical for reducing background and ensuring signal specificity.

Step 6: Detection & Analysis

Radioactive: Wrap the blot in saran wrap and expose it to a phosphorimager screen. Exposure times can vary from 30 minutes to several days, depending on signal strength.
Non-Radioactive: If using biotin-labeled probes, proceed with a detection protocol involving streptavidin-HRP conjugates and a chemiluminescent substrate (e.g., BrightStar BioDetect Kit).

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Northern Blot FAQs & Troubleshooting

Can I detect multiple RNAs on one blot?

Yes. The best practice is to design specific probes for each RNA of interest and then mix the probes together in the hybridization buffer. If the RNAs are different sizes (e.g., a 21 nt miRNA and a 24 nt siRNA), they will appear as distinct bands. This is common for detecting related family members, such as the let-7 miRNA family.

Why is my signal weak?

The most likely culprit is poor RNA retention. Ensure you are using a positively charged nylon membrane and, most importantly, switch from UV cross-linking to the far more sensitive EDC chemical cross-linking method.

Does a northern blot detect RNA?

Yes, the Northern blot is the classic and definitive technique used specifically for the detection of RNA. It allows researchers to separate RNA molecules by size and then use a complementary probe to confirm the identity, size, and relative abundance of a specific RNA transcript within a complex sample.

How do you identify small RNA?

Small RNAs (like miRNAs, siRNAs, and piRNAs) are identified using a specialized, high-resolution Northern blot protocol. The key steps are:

Separation: Total RNA is run on a high-percentage denaturing polyacrylamide gel (Urea-PAGE), which is necessary to resolve these very small <40 nucleotide transcripts.
Transfer: The RNA is transferred to a positively charged nylon membrane.
Immobilization: The RNA is permanently fixed to the membrane. For small RNAs, EDC chemical cross-linking is vastly superior to standard UV cross-linking and provides a much stronger signal.
Hybridization: A specific, labeled oligonucleotide probe (often 32P-labeled for high sensitivity) that is complementary to the small RNA sequence is incubated with the membrane.
Detection: The probe's signal is visualized, revealing a band at the precise molecular weight of the small RNA.

What is the limit of detection for northern blot?

The limit of detection varies significantly based on the protocol, probe type, and the abundance of the target RNA. For small RNAs, which are notoriously difficult to retain on the membrane, traditional UV-cross-linking protocols have a poor detection limit.

However, the optimized protocol outlined in our article, which uses EDC chemical cross-linking, has been shown to increase signal detection by up to 50-fold. When combined with a high-specific-activity radioactive [γ-32P]ATP probe, this method is extremely sensitive and is considered the "gold standard" for validating even low-abundance small RNAs.

What kind of blot is used to detect RNA?

The Northern blot is the specific technique used to detect RNA. This method is named in contrast to its precursor, the Southern blot, which is used to detect DNA. Likewise, a Western blot is used to detect proteins.