Method of the Month — The Northern Blot
October 2021
Next in our series of “gel-based” methods is Northern Blotting, a technique that allows for the detection of specific RNA molecules in a sample. Make sure to also read last month’s discussion of Western Blotting, as well as this basic outline of gel electrophoresis.
Imagine that you are a biologist working with a tissue sample, and you want to study gene expression by checking for a specific molecule of RNA. We will call him Richard.
In this cluster of cells, you realize that there are easily millions of RNA molecules floating around, but most of them are irrelevant to you. You want to find Richard, but where are you to begin with this mess of nucleotides?
The answer to our predicament is to use a gel-based method known as Northern Blotting. What is this method, and where are we to begin on our quest to isolate Richard?
First, we must denature, or separate the RNA within our sample into single, unfolded strands. This can be done in a variety of ways. One way is through the chemical modification of guanine to disrupt secondary structure. In the past, this was done using formaldehyde or methyl mercury, but modern labs can carry out this step much more safely by using buffer solutions. As you may recall, guanine is a base pair of RNA that is complementary to cytosine. If the structure of guanine is altered, it will no longer be able to bind to cytosine, which will ensure that all fragments of RNA are single stranded and able to be separated in the next step.
Next, we must use gel electrophoresis to separate the RNA fragments according to size. For a more in-depth explanation of gel electrophoresis, click here. If you need a quick refresher on this method, an electric current is applied to a gel which causes RNA fragments of varied sizes to migrate to different areas of the gel. Smaller fragments will appear at the bottom, while larger fragments will be closer to the top.
Then, we transfer this separated RNA onto a sheet of nitrocellulose membrane. To conduct this transfer, we will construct a blotting sandwich. The layers of the blotting sandwich are shown below.
The final product of this step will be a blotting membrane displaying all the RNA bands from the original gel. The use of this blotting membrane is where the Northern blot gets its name!
We will then treat this membrane with a small piece of DNA known as a probe, which is designed to complimentarily bind to a specific target RNA fragment. In our case, we will use a probe that is specific to Richard’s sequence and will bind to him once it finds him. We will call her Rachel.
But what about all the probes that are bound nonspecifically, won’t they dull Richard’s shine? Fear not, we will simply wash them away with a substance known as SSPE buffer, then dry and analyze our result. Now, not only will we be able to tell if Richard was in our sample, but we will also be able to see approximately how many nucleotides long he is. To see actual images of completed Northern Blots, check out this article.
As you can see, we have completed our Northern Blot! We first denatured the RNA within a sample, then used gel electrophoresis to separate these RNA molecules according to size. After that, we transferred the RNA from a gel to a nitrocellulose membrane and treated it with a probe. This allowed us to visualize the target gene of our sample. We hope you enjoyed this feature, and we will see you next month for our next gel-based method!
