Northern blot is a molecular biology technique used to detect levels of RNA molecules of a specific sequence in your sample. This method is useful for monitoring the transcription and transcript abundance of a target gene in a cell or tissue sample by assaying for the corresponding RNA.
By measuring the transcript levels of your gene of interest, you can monitor the tissue-wide expression pattern of your gene as well as how its transcript is turned over. And in addition to detecting the quantity of specific transcripts, Northern blot also helps determine the sizes of specific transcripts under investigation.
This technique, developed in 1977, is similar to Southern blot, but it analyzes different nucleic acids. While Southern blot analyzes DNA, Northern blot analyzes RNA.
Article table of contents:
General steps in a Northern blotting experiment
Northern blotting applications
General steps in a Northern blotting experiment
The Northern blotting technique involves separating RNA molecules based on their size by agarose gel electrophoresis (transcript size >=1kb) or polyacrylamide gel electrophoresis (transcript size < 1kb).
The gel is then transferred to a membrane, such as nitrocellulose or nylon, where RNA is immobilized.
Following the transfer, RNA is hybridized with a DNA or RNA probe whose sequence complements the target RNA molecule. This enables researchers to detect the RNA molecule by autoradiography or other detection methods.
Let’s take a closer look at the Northern blotting steps in detail.
RNA extraction
The initial and crucial step in the Northern blot technique is RNA extraction, which determines the quality and quantity of the RNA molecule being analyzed.
Two methods can be used to extract RNA from cells or tissues:
The first method involves organic solvents like phenol and chloroform to extract RNA. The RNA sample, from leaf tissue, for example, is homogenized using guanidium isothiocyanate and phenol which disrupts cells and inactivates RNases. RNA is then separated by chloroform and precipitated with isopropanol.
This method is efficient, fast, and provides high-quality RNA for the downstream procedure.
The second method involves a silica-based column. This method is more expensive, especially when you are extracting RNA from multiple samples.
In both methods, the extracted RNA is treated with DNases to eliminate any contaminating DNA.
The quality and quantity of the extracted RNA can be assessed using a spectrophotometer which measures the concentration, purity, and integrity of your sample.
You can also do a simultaneous electrophoresis of your RNA to confirm that your extracted RNA is not degraded.
Something important to keep in mind with this is that it is essential to handle RNA samples carefully because RNAs are susceptible to degradation by RNases.
RNases are ubiquitous enzymes that degrade RNA. You must wear RNase-free gloves because your own fingers can be a source of RNase.
Avoid RNase contamination by using RNase-free reagents, equipment, and work surfaces, and sterilize all equipment and work surfaces.
mRNA isolation
Total RNA extracted from a sample will include all the different types of RNA such as messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and other non-coding RNA.
However, if the focus of the experiment is to study gene expression changes, then it is desirable to isolate mRNA specifically because it represents the subset of RNA molecules that are translated into proteins.
To isolate mRNA from total RNA, a poly-A (+) selection procedure is typically used.
An oligo-dT column or beads primed with oligo-dT is used for this procedure. Oligo-dT selectively binds to the poly-A tail of mRNA, which is a common feature of most eukaryotic mRNA.
The non-mRNA components of the RNA sample that do not contain a poly-A tail will not bind to the oligo-dT column or beads, leading to their selective removal.
The isolation of mRNA from other RNA molecules increases the sensitivity of downstream applications, as it removes most of the non-coding RNA and rRNA which can interfere with mRNA analysis.
RNA separation
Once you have your extracted total RNA sample or the isolated mRNA, you need to separate the RNA molecules based on size.
Separating RNA molecules from each other helps with downstream identification and target RNA analysis, giving you much higher resolution.
For this, the extracted RNA is run on an agarose or polyacrylamide gel. Formaldehyde is added to the gel to prevent formation of RNA secondary structures. Once the gel is run, you can visualize your separated RNA fragments by staining the gel using ethidium bromide (EtBr) or an alternative like https://goldbio.com/search?q=gelredhttps://goldbio.com/search?q=gelred under UV light.
Now let’s get into the more prominent steps of Northern blot, and that starts with RNA transfer from the gel to a membrane.
RNA transfer
RNA is transferred from the gel to a membrane, such as nitrocellulose or nylon.
A nylon membrane is generally preferred over nitrocellulose because of its high affinity toward nucleic acid molecules. The positive charge within the nylon membrane binds to the negatively charged RNA molecules with great robustness.
There are two methods that aid the transfer process for blotting: capillary blotting and vacuum blotting.
Capillary blotting is the traditional approach taken by scientists where RNA transfer from the gel to the membrane occurs by capillary action. This method is simple and requires no special equipment, but the transfer process is pretty slow.
Alternatively, vacuum blotting is becoming increasingly popular because it offers several advantages over traditional capillary blotting.
This method uses a vacuum pump to transfer the RNA molecules from the gel to the membrane, which is faster, taking only 1-2 hours, and improves reproducibility.
The choice of blotting method depends on the specific requirements of the experiment. Capillary blotting is a good choice when you are working with only a few different RNA samples, and time is not a significant constraint.
However, vacuum blotting comes in handy when a large number, for example, 50 different RNA samples need to be processed quickly and with higher reproducibility.
After the transfer process using either capillary or vacuum blotting, the RNA molecules are immobilized on the membrane through covalent linkage. This linkage is achieved by exposing the membrane to 254 nm UV light or by heating it to around ~80°C.
It is crucial to immobilize RNA molecules to avoid losing them during the subsequent processing steps. Failure to cross-link the RNA properly could compromise the assay's accuracy and sensitivity.
Pre-hybridization (blocking)
To avoid a reduced signal-to-noise ratio caused by high background levels on the membrane, it is important to perform thorough washes to minimize any non-specific binding with the membrane or other RNA molecules.
To ensure that the probe used in the subsequent hybridization step interacts solely with the mRNA of interest, a blocking agent (such as salmon sperm DNA) is used.
