Azure BiosystemsNorthern blotting is a technique used by molecular biologists to detect a specific RNA sequence in a sample. RNA plays many roles in the cell. The most well-known type of RNA, messenger RNA (mRNA), is transcribed from genes to provide the templates used by ribosomes to synthesize proteins via translation. Northern blotting has traditionally been used to measure mRNAs to study gene expression. However, this type of analysis has also been applied to other RNAs such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs).
A challenge associated with Northern blotting is that RNA degrades quickly due to RNases either in the sample itself or from environmental contamination. In order to be successful with this method, it is essential to use RNase-free reagents, sterilize all glassware, and clean all surfaces with RNase inhibitors. Despite the multiple steps involved and the extra care required for success, Northern blotting remains an important technique for studying RNA.
Northern blots are often performed to analyze gene expression of one or a small number of genes. An advantage of using this type of analysis over other RNA analysis techniques is it visualizes the size of RNA molecules which can reveal differences in processing, such as splicing variants.
In addition, Northern blotting can be quantitative to allow comparison of expression levels between samples. Therefore, it is often used to confirm gene expression data obtained from high-throughput gene expression studies, such as microarrays or real-time PCR.
The probes used on Northern blots are small RNA or DNA nucleotides. Traditionally, probes are labeled with radioisotopes, particularly 32P, and detected using X-ray film or phosphor imaging on an instrument such as the new Azure Sapphire. Using radiolabeled probes allows for quantitation of RNA species so that expression levels can be compared between samples. However, 32P has a short half-life, so labeled probes cannot be stored and used in future experiments.
Researchers may wish to avoid working with radioactive reagents due to additional complications and requirements associated with storing, handling, and disposing of radioactive chemicals. Therefore, non-radioactive alternative methods for probing Northern blots have become popular.
A commonly used non-radioactive detection method for Northern blotting is to label the probe with digoxigenin or biotin. The probe is indirectly detected using an anti-digoxigenin antibody or streptavidin labeled with HRP (for subsequent chemiluminescent detection) or a fluorophore. Alternatively, probes incorporating near-infrared (NIR) fluorophores have been used to directly detect the target RNA. Many protocol modifications have been published to optimize the sensitivity depending on the label used and the size of the target RNA.
The new Azure Sapphire FL Biomolecular Imager is a perfect choice for imaging Northern blots. It is an instrument that can carry out phosphor imaging for use with radiolabeled probes. It can also detect NIR fluorescence and is compatible with newer fluorescent detection methods. The Sapphire FL has the option to add a Chemiluminescence module to allow you to perform chemiluminescence imaging when using digoxigenin or biotin-labeled probes detected with HRP-conjugated secondary antibodies or streptavidin.
Other options for imaging Northern blots include either the Azure 500 or Azure 600 Imagers; both of which are also excellent choices for imaging your blots with chemiluminescence or NIR fluorescence.
It is a technique used to detect RNA sequences in a sample.
Northern blotting is used to detect specific RNA sequences and measure how much of an RNA molecule is present. In addition, you can also use it to study gene expression.
Northern blots detect RNA, while Southern blots detect DNA.
Laser-customizable imaging system that carries out phosphor imaging for use with radiolabeled probes
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Moritz, CP. 40 years Western blotting: A scientific birthday toast. Journal of Proteomics. Vol 212. 2020.
New insight into microtubule-kinetochore interactions Work recently published in the Journal of Cell Biology1 provides new insight into a fundamental processes of cell biology, chromosome segregation. Flores et al
We can’t think of a better way to start off our blog posts with a post covering the history of Western blotting and bust a
