Week 4: Quality Over Quantity
Have you ever heard the quote “You only get out of life what you put in”?
Well, the same is true for RNA. The quality of RNA you sequence, determines the quality and accuracy of the data generated from sequencing. In the last post, we discussed my steps in obtaining RNA of a high purity, in other words RNA only minimally contaminated by proteins, DNA or other extraction chemicals.
However, what hasn’t been discussed yet is the quality of the RNA. Quality and purity might sound like the same thing, but quality refers to the integrity of the nucleic acid themselves while purity refers to contamination.
To determine the quality of my extracted RNA, I performed RNA gel electrophoresis.
Gel electrophoresis is a process by which you can separate samples of nucleic acids by their molecular weight. Because nucleic acids are negatively charged, applying a current to them will cause the RNA to migrate through an agarose gel matrix. However, the speed at which the RNA travels through the gel varies depending on the length (molecular weight) of the RNA. The smaller the RNA, the faster and farther it will move through the gel. In the end, there will be distinct bands of RNA separated by their molecular weight. If the bands are smeared, it’s highly likely that the RNA is degraded. Furthermore, if we see a band near the well, that would indicate possible high molecular weight genomic DNA contamination. Below is a picture of a gel electrophoresis setup.
Here are the steps to perform gel electrophoresis.
- Prepare an 1% agarose gel by adding 1g of molecular grade agarose to 1X TAE (Tris Acetate EDTA) buffer
- Heat the solution until the agarose fully dissolves.
- Allow the gel solution to cool to 60°C.
- Add a fluorescent nucleic acid visualization dye to the solution and swirl to mix.
- Pour the gel solution into a gel casting tray with a comb to create wells.
- Fill the electrophoresis chamber with TAE buffer.
- Remove the comb and place the gel in the chamber.
- Add a loading buffer to the RNA sample.
- Load the RNA sample into the wells of the gel.
- Turn on the current and wait 30 minutes for the RNA sample to migrate.
- Visualize the RNA samples under a blue light illuminator as seen below.
I ran 120 nanograms of my RNA through the gel for approximately 45 minutes. The fluorescent dye we added while preparing the agarose gel binds to the RNA and is excited by the blue light illuminator revealing the image below.
In the image above, the left sample is the RNA, and on the right is a positive control ladder. We can see that there are two distinct bands of RNA with minimal smearing. The top most band corresponds to 28S Ribosomal RNA and the band below is corresponds to the 18S Ribosomal RNA. Furthermore, the 28S band is brighter than the 18S band. These observations match up with prior literature on eukaryotic RNA indicating that high quality RNA was in fact obtained.
Thank you again for taking the time to read my blog post! Make sure to leave aby comments below if you have questions. Stay tuned for my next one!