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Southern Blot

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...

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Updated: Nov 18, 2025

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

Published on: July 11, 2020

R-Loop Analysis by Dot-Blot.

Prisila Ramirez¹, Robert J Crouch², Vivian G Cheung³

¹Life Sciences Institute, University of Michigan, Ann Arbor.

Journal of Visualized Experiments : Jove

|February 8, 2021

Summary

This summary is machine-generated.

R-loops, crucial for gene regulation, can now be reliably quantified using a sensitive dot-blot assay. This method validates R-loop detection and aids in studying their role in human cells and diseases.

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

R-loops are three-stranded nucleic acid structures with significant roles in gene regulation.
Previously considered transcription by-products, R-loops are now known to have functional importance in human cells.
Understanding R-loop abundance and cellular regulation is critical, especially given their altered levels in disease states.

Purpose of the Study:

To develop and validate a sensitive and reproducible method for quantifying R-loops.
To address the specificity concerns surrounding the commonly used S9.6 antibody for RNA-DNA hybrid detection.
To establish an assay applicable to both research and clinical settings for R-loop abundance assessment.

Main Methods:

Utilized dot-blots employing the S9.6 monoclonal antibody for R-loop quantification.
Employed RNase H, RNase T1, and RNase III to confirm the assay's specificity for RNA-DNA hybrids and different RNA forms.
Validated the assay's sensitivity, reproducibility, and ease of use with standard laboratory equipment.

Main Results:

Demonstrated the sensitivity and specificity of the S9.6 antibody-based dot-blot assay for quantifying R-loops.
Confirmed that RNase H specifically degrades RNA-DNA hybrids, validating the assay's target detection.
Showcased the assay's high reproducibility and ability to yield results within two days.

Conclusions:

The developed dot-blot assay provides a reliable method for R-loop quantitation.
This assay overcomes previous specificity challenges and can be used to study R-loop dynamics.
The method is suitable for assessing R-loop levels in research and clinical contexts, including the impact of genetic mutations like those in senataxin.