Competitive dot blot protocol

Last edited Tue 1 Jun 2025

Introduction to RNA analysis

RNA analysis is fundamental to understanding the complex roles that the numerous types of RNA molecules play in cellular processes, gene expression, and regulation. By examining how RNA interacts with other molecules, especially RNA-binding proteins, researchers can uncover the mechanisms that control the fate and function of RNA within the cell. These RNA-binding proteins recognize and bind to specific target RNA sequences, influencing everything from RNA stability to translation and splicing. Techniques such as dot blot and northern blot are widely used to study these interactions, allowing scientists to assess the binding affinity and specificity of proteins for their target RNA. Through these methods, researchers gain valuable insights into how gene expression is regulated at the RNA level, helping to unravel the intricate networks that govern cellular function.

Dot blot protocol overview

The dot blot protocol is a straightforward and versatile method for detecting and analyzing interactions between RNA and proteins. In this approach, RNA samples are immobilized onto a membrane, commonly a nitrocellulose or PVDF membrane, creating a stable platform for probing. After the RNA is fixed to the membrane, it is incubated with a specific antibody or probe designed to detect a particular RNA-binding protein. The protocol involves several key steps: preparing the RNA sample, preparing and marking the membrane, applying the RNA to the membrane (dot blotting), hybridizing with the specific antibody, and finally, detecting the bound protein. This method is highly effective for studying RNA-protein interactions, as it allows for the rapid and sensitive detection of binding events between RNA samples and their corresponding proteins. The dot blot protocol is valued for its simplicity, efficiency, and adaptability to a wide range of experimental needs.

Applications of dot blot

The dot blot protocol is a powerful tool in RNA research, offering a range of applications for studying the molecular interactions that drive cellular processes. One of its primary uses is in the investigation of RNA-protein interactions, enabling researchers to identify and characterize RNA-binding proteins and their binding affinity for specific target RNA sequences. This technique is also instrumental in exploring RNA structure, as it can reveal how secondary and tertiary structures influence protein binding. In addition, dot blot assays are frequently used to monitor gene expression by detecting the presence and abundance of specific RNA molecules in cells or tissues. The protocol’s versatility extends to studying interactions between RNA and other nucleic acids or proteins, making it a valuable method for analyzing the specificity and dynamics of molecular binding events. Whether examining changes in gene expression or mapping the landscape of RNA-protein interactions, the dot blot protocol provides reliable and sensitive detection for a variety of research applications.

Competitive dot blot vs. dot blot: Key differences

A competitive dot blot is a quantitative immunoassay designed to measure the concentration of a target antigen by leveraging competition between a known quantity of labeled antigens vs. unlabeled antigens. Unlike a standard dot blot, which directly detects antigen presence on a membrane, the competitive format involves pre-incubating a fixed amount of a labeled antigen with varying concentrations of unlabeled sample antigen and a limited amount of antibody. This mixture is then applied to a membrane. The more unlabeled antigen present, the less labeled antigen binds to the antibody, resulting in a weaker signal. Signal intensity is inversely proportional to antigen concentration, and a standard curve is typically used for quantification. This makes competitive dot blot ideal for sensitive detection of small molecules or modified nucleosides where direct detection is challenging. In contrast, traditional dot blots are more qualitative or semi-quantitative and do not involve competitive binding.

Best practices and safety precautions

To achieve accurate and reproducible results with the dot blot protocol, it is essential to follow best practices and observe safety precautions throughout the experiment. Handle all RNA samples with care to prevent degradation, use RNase-free reagents and equipment, and keep samples on ice whenever possible. When preparing membranes and performing hybridization, ensure that the concentration of the RNA sample and the hybridization conditions are optimized for your target RNA sequence to maximize detection sensitivity and specificity. Take special care when working with hazardous materials such as ethidium bromide and when using UV light for crosslinking, following all laboratory safety guidelines to protect yourself and your samples. Proper membrane handling, including avoiding direct contact with pipette tips and ensuring even sample application, will help minimize background noise and improve detection accuracy. By carefully controlling experimental conditions and adhering to safety protocols, you can ensure reliable detection of your target RNA and gain meaningful insights into RNA structure, function, and molecular interactions.

Stage 1 - Procedure

Materials required

Reagents

• RNA samples or oligos
• RNase-free water
• Wash buffer, eg TBST (1x TBS, 0.1% Tween-20)
• Blocking Solution
• Primary antibody
• Secondary antibody (goat anti-rabbit IgG-HRP, ab97051, or goat anti-mouse IgG-HRP)
• ECL substrate
• Nucleoside competitors
• Streptavidin-AlexaFluor488
• Methylene blue staining buffer (ab246830) (0.2% Methylene blue in 0.4M sodium acetate and 0.4M acetic acid)

Equipment

• RNase-free tubes
• Heat block
• Positively charged nylon membrane
• 10 cm plastic petri dish
• SG Linker (with 254 nM bulb)
• Imaging system

Steps

Dilute the RNA samples or oligos containing the modification of interest to an appropriate concentration with RNase free water.

Denature the diluted RNA sample at 95 degrees Celsius in a heat block to disrupt secondary structures for 3 min.

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

Cut the positively-charged membrane to an appropriate size.

• Mark a 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 and drop 1 µL of RNA onto the positively charged nylon membrane.

Transfer the dish with the membrane immediately into the chamber of SG Linker.

• Remove the dish lid and crosslink the RNA to the membrane (RNA side up) with UV light: 125 mJoule/cm² at 254 nM.

Cut the membrane according to the grid and transfer to a clean 24-well plate.

Wash the membrane in 500 μl of wash buffer for 5 min at room temperature with gentle shaking to wash off the unbound RNA.

Incubate the membrane (RNA side down) in 300 μl blocking buffer for 1 h at room temperature with gentle shaking.

Wash the membrane in wash buffer for 10 min.

Dilute the nucleoside competitors serially in blocking buffer to give final concentrations of 1,000/200/40/8/0 nM.

• Incubate each dilution with 1 μg/ml of the antibody you are testing.

Incubate the membrane with the antibody in 300 μl blocking buffer overnight at 4 degrees Celsius with gentle shaking.

Wash the membrane (flip the membrane so the RNA side faces up) three times for 10 min each in 10 ml of wash buffer with shaking.

Incubate the membrane (RNA side down) with an appropriate secondary IgG-HRP antibody diluted in blocking buffer for 1 h at room temperature with gentle shaking.

Wash the membrane (RNA side up) four times for 10 min each in 10 ml of wash buffer with shaking.

Incubate the membrane with ECL substrate (eg ab133406) for 5 min.

Expose the membrane with your imaging system and take images in high-resolution mode from 1 s exposure up to 3 min exposure with appropriate intervals.

Wash the membrane in wash buffer for 30 min.

• Incubate the membrane with Streptavidin-AlexaFluor488 (1:5,000 in wash buffer) for 1 h in a black box
• Expose the membrane for Alexa 488 (Ex/Em: 490/525 nm) with your imaging system.

After fluorescence imaging, transfer the membrane to a petri dish containing 10 ml Methylene blue staining buffer.

• 10 ml Methylene blue staining buffer should be 0.2% Methylene blue in 0.4 M sodium acetate and 0.4 M acetic acid.
• Incubate the membrane for 30 min with gentle shaking.

Rinse the membrane with tap water until the background is clean (around 30 s to 60 s wash).

Take a photo of the methylene blue stained membrane with your imaging system.