RNA dot blot protocol

Last edited Tue 01 July 2025

RNA dot blot is a technique similar to northern blotting; however, RNA samples are not separated by electrophoresis. Instead, they are spotted onto a membrane.

Introduction to RNA dot blot

RNA dot blot is a powerful molecular biology technique designed to detect and quantify specific RNA sequences within a variety of RNA samples. Unlike traditional northern blotting, which separates RNA by size, the dot blot method involves directly spotting RNA samples onto a positively charged nylon membrane. After immobilization, a labeled probe, complementary to the target RNA sequence, is used to reveal the presence and abundance of specific transcripts. This approach is widely used for gene expression studies, enabling researchers to compare gene activity across different tissues, cell types, or experimental conditions. The use of a nylon membrane ensures efficient binding of nucleic acids, while the rapid and parallel processing of multiple samples makes RNA dot blot an essential tool in molecular biology research.

Preparation of materials and equipment

Setting up a successful dot blot assay requires careful preparation of both materials and equipment. Essential components include a positively charged nylon membrane for optimal nucleic acid binding, a dot blot apparatus for precise sample application, and high-quality RNA samples, often total RNA extracted from cells or tissues. Labeled probes, specifically designed to hybridize with the target RNA, are prepared for specific detection. The hybridization solution facilitates the binding of the probe to the RNA, while wash buffer and blocking buffer are critical for minimizing background signals and ensuring specificity. Proper use of blocking buffer helps prevent non-specific interactions on the nylon membrane, and thorough washing with wash buffer removes unbound probes, resulting in clear, interpretable results. Each step, from sample preparation to membrane handling, is crucial for the accuracy and reliability of the dot blot.

Stage 1 - Procedure

Materials required

Reagents

• RNA sample
• RNase-free water
• TBST (1x TBS, 0.1% Tween-20)
• Blocking solution (AdvanBlock-Chemi Blocking solution (R-03726-E10, Advansta))
• Primary antibody
• Secondary antibody (goat anti-rabbit IgG-HRP, ab97051, or goat anti-mouse IgG-HRP)
• ECL substrate
• Streptavidin-AlexaFluor^® 488
• Methylene blue staining buffer (0.2% Methylene blue in 0.4M sodium acetate and 0.4M acetic acid)​

Equipment

• RNase-free tubes
• Heat block
• Positively charged nylon membrane (Hybond-N ⁺ membrane (Amersham RPN303B, GE))
• 10 cm plastic petri dish
• SG Linker (with 254 nM bulb)
• Imaging system​

Alexa Fluor^® is a registered trademark of Life Technologies. Alexa Fluor^® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

Steps

Take the RNA sample (aliquoted) from the freezer. Thaw the RNA sample tube by hand and put back on ice.

Using RNase-free tubes, serially dilute the RNA sample with RNase-free water.

• We use final concentrations of 2.5/0.25/0.025/0.0025 μg/μL.
• We dilute the positive oligo containing the RNA mod and negative oligos without modifying interest into both 50 ng/μL and 5 ng/μL.

Incubate the serially diluted RNA at 95°C in a heat block for 3 min to disrupt secondary structures.

Chill the tubes on ice immediately after denaturation to prevent the re-formation of secondary structures of RNA.

Cut the positively charged nylon membrane to an appropriate size, it needs to be big enough for a 2 X 4 grid.

• Mark the grid on the membrane lightly with a pencil to guide sample loading.
• Transfer the membrane to a clean 10 cm plastic petri dish.

Mix the RNA sample by pipetting up and down. Drop 2 µL of RNA onto the membrane.

Transfer the dish with membrane immediately into the chamber of SG Linker (with 254 nM bulb).

• Remove the dish lid and ensure the membrane is RNA side up.
• Crosslink RNA to the membrane with UV light: 125 m Joule/cm² at 254 nM.

Wash the membrane in 10 mL of TBST (1X TBS, 0.1% Tween-20), for 5 min at room temperature with gentle shaking to wash off the unbound RNA.

Incubate the membrane (RNA-side down to prevent accidental drying out of the membrane) in 10 ml blocking buffer for 1 h at room temperature with gentle shaking.

Discard the blocking buffer, incubate the membrane with primary antibody (at given dilution, usually 1 μg/mL working IgG) in 10 ml blocking buffer overnight at +4°C with gentle shaking.

Flip the membrane so it is RNA-side up.

• Wash the membrane three times for 10 min each in 10 mL of TBST with gentle shaking.

Flip the membrane so it is RNA-side down.

• Incubate the membrane with appropriate secondary antibody (goat anti-rabbit IgG-HRP (1:20,000 dilution, ab97051) or anti-mouse IgG-HRP) in blocking buffer for 1 h at room temperature with gentle shaking.

Flip the membrane so it is RNA-side up.

• Wash the membrane four times for 10 min each in 10 mL of TBST with gentle shaking.

Incubate the membrane with ECL substrate for 5 min.

Expose the membrane with your imaging system.

• Take the image on high-resolution mode from 1 s exposure up to 3 min exposure with appropriate intervals. After exposure, wash the membrane in TBST for 10 min.

Incubate the membrane with Streptavidin-AlexaFluor^® 488 (1:5000 in TBST) for 1 h in a black box.

Transfer the membrane to your imaging system.

• Take the image with Alexa 488 fluorescence mode.

After fluorescence imaging, transfer the membrane to a petri dish containing 10 mL methylene blue staining buffer (0.2% methylene blue in 0.4M sodium acetate and 0.4M acetic acid).

• Incubate the membrane for 30 min with gentle shaking.

Wash the membrane with dH₂O until the background is clean (around 30 s to 60 s).

Image the methylene blue-stained membrane with your imaging system.

Data analysis and interpretation

After completing the dot blot assay, the next step is to analyze the data obtained from the nylon membrane. Signal intensity from the labeled probe is measured, often using an imaging system, and this reflects the amount of target RNA present in each sample. To ensure accurate comparisons, signal intensity is typically normalized to an internal control, such as a housekeeping gene, which accounts for variations in sample loading and hybridization efficiency. This normalization allows for the quantitative assessment of gene expression and the identification of differential expression patterns across samples or conditions. By comparing these patterns, researchers gain valuable insights into the regulation of gene expression and its impact on various biological processes. Careful analysis of the dot blot results is essential for drawing meaningful conclusions from the experiment.

Troubleshooting and limitations

While RNA dot blot is a robust technique, several challenges can affect the quality and reliability of results. RNA degradation is a common issue, so it is vital to use RNase-free reagents and handle RNA samples with care to preserve nucleic acid integrity. Incomplete denaturation of RNA can lead to secondary structures that hinder probe binding, while non-specific binding of the labeled probe to the nylon membrane can increase background signals. To address these issues, optimize hybridization and washing conditions, and always include appropriate controls, such as a negative control and an internal control, to validate the assay. By following best practices and troubleshooting as needed, researchers can minimize errors and ensure the accuracy of their dot blot experiments.

Applications

RNA dot blot assays are widely used in molecular biology for a range of applications, including gene expression analysis, studies of RNA modifications, and diagnostic testing. This technique enables researchers to examine gene expression patterns in different tissues, cell types, or under various experimental conditions, providing insights into the regulation of several biological processes. RNA dot blot can be combined with other methods, such as northern blot analysis, RT-PCR, and blot analysis, to offer a more comprehensive view of gene regulation. Looking ahead, advances in labeling technologies and the integration of dot blot with genomics and proteomics approaches promise to expand its utility, enabling deeper exploration of molecular medicine and complex biological systems.