How do you know if your RNA is good enough for downstream analysis?

An easy, affordable way to verify the RNA quality is through agarose gel. Keep in mind, gel electrophoresis can show different results that need interpretation. Fortunately, there are some principles that once understood, help this process out.

This article covers the principles for RNA integrity and how those principles are interpreted in RNA gel electrophoresis.


Framing interpretation: Necessary RNA overview

In order to properly conduct RNA gel interpretations, it’s important to get a good refresh about the types of RNA present in cells and their structure.

There are three main types of RNA:

  • Messenger RNA (mRNA): Messenger RNA, or mRNA, is a molecule that holds the DNA sequence and encodes for proteins or regulation products.
  • Ribosomal RNA (rRNA): Ribosomal RNA, or rRNA, is part of the ribosome structure and is essential for protein synthesis in all living organisms. rRNA constitutes the primary material within the ribosome, approximately 60% rRNA and 40% protein by weight.
  • Transfer RNA (tRNA):Transfer RNA, or tRNA, is a type of RNA molecule that contributes to decoding the mRNA sequence into a protein. Transfer RNAs function at specific sites in the ribosome during translation (the process that synthesizes a protein from an mRNA molecule).

The proportion of these three types of RNA varies in each cell. However, rRNA occupies 80% or more, followed by tRNA with 14% and ending with mRNA at about 2.5%.

Chart showing the three types of RNA, their abundance and function - rRNA, tRNA, and mRNA

Researchers use rRNA as an indirect reference to measure the mRNA quality because of is high abundance in cells.

Before explaining how the rRNA is used as a reference, we need to look at rRNA structure in more detail.


rRNA as a quality reference

Ribosomal RNA has two subunits called small and large subunits.

Each subunit is composed of RNA and proteins. For each subunit, the proportion of RNA and proteins varies. Furthermore, the rRNA differs in prokaryotes and eukaryotes.

In prokaryotes, the small subunit has two rRNAs called 23S rRNA and 5S rRNA and 31 proteins, while the large subunit has one rRNA called the 16S rRNA plus 21 proteins.

In eukaryotes, the small subunit comprises three rRNAs called 5S rRNA, 5.8S rRNA, and 25/28S rRNA plus 50 proteins. The large subunit has one rRNA called the 18S rRNA and 33 proteins.

Animals and plants only differ in the small subunit, where 25-26S is common in plants, and 28S rRNAs occurs in animals.

Here it is essential to clarify that the assembly of the subunits is not additive, meaning that when the small and large subunits in prokaryotes merge the 50S and 30S does not sum to 80S. Instead, it is 70S.

The same can be said for eukaryotes, where 60S and 40S subunits assembly gives a ribosome of 80S rather than a sum of 100S.


Why ribosomal subunits don’t add up

Ribosomal subunits are classified as per sedimentation rate. The sedimentation rate for a particle of a given size and shape measures how fast the particle "settles."

Here, the "S" in the rRNA means the Svedberg's unit, which measures the sedimentation rate.

Experiments show that when each subunit is sedimented individually, the corresponding values are 50S/30S in prokaryotes and 60S/40S in eukaryotes for large and small subunits, respectively.

However, when small and large subunits are sedimented together, the overall sedimentation rate turns out to be 70S in prokaryotes and 80S in eukaryotes.

chart showing RNA subunit size


Interpreting the RNA quality on a gel

RNA quality is essential for downstream analysis for clear and accurate conclusions.

To clarify, when discussing RNA quality, we are talking about its integrity.

When it comes to RNA integrity, it is important to remember the amount of RNA types within cells: rRNA > tRNA > mRNA.

In general, most of researchers want to sequence and study the mRNA because mRNA is the molecule being translated into proteins. So, scientists study gene expression by studying the mRNA.

However, due to the low proportion of mRNA within cells, results and mRNA quality analysis can be difficult to achieve directly

Instead, researchers use rRNA as a quality reference. Researchers expect to find in their RNA gels two bands, each corresponding to one subunit of rRNA. Then, the hypothesis is, if rRNA subunits look ok and are not smeared, mRNA will also be intact.

RNA electrophoresis gel rendering


One of the most affordable and quickest ways to verify the RNA quality is by performing an RNA gel electrophoresis.

And when it comes to interpreting RNA gel electrophoresis results, there are two scenarios we should walk through.


Scenario 1: Bad RNA Quality

One situation you might encounter while doing RNA electrophoresis is bad RNA quality.

Here you may find two smeared bands, only one smeared band, or no bands.

Having smeared bands or only one band usually corresponds to degraded RNA while having no bands corresponds to the absence or RNA or insufficient RNA content.

Figure shows different scenarios of bad RNA quality in an agarose gel and causes of problem


Scenario 2: Good RNA Quality

The second, and ideal situation you may encounter is getting good quality RNA.

When you have good quality RNA, you will clearly identify two complete bands. Other researchers call it, complete RNA.

  • Prokaryotes: for prokaryotes (e.g., bacteria), you will clearly see 23S and 16S RNA bands.
  • Plants: For plants, good quality RNA will allow you to easily see 25S and 18S RNA bands.
  • Mammals: If you work with mammals, you will clearly see 28S and 18S bands on your RNA gel.

Diagram showing scenarios of good RNA quality and subunit sizes for each model type

Remember that on the gel you will only detect the RNA portion of the ribosome and in general you may detect only the larger RNA portion within the small subunit.



figure showing evaluation of good quality rna results in an agarose gel


Keywords:

RNA integrity, gel electrophoresis, RNA types, ribosomal RNA.




References

Aranda, P. S., LaJoie, D. M., & Jorcyk, C. L. (2012). Bleach Gel: A Simple Agarose Gel for Analyzing RNA Quality. NAtional Institutes Health, 33(2), 366–369.

Chacko, S. (2005). Validating RNA quantity and quality: Analysis of RNA yield, integrity, and purity. BioPharm International.

Imbeaud, S., Graudens, E., Boulanger, V., Barlet, X., Zaborski, P., Eveno, E., Mueller, O., Schroeder, A., & Auffray, C. (2005). Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Research, 33(6), 1–12.

Tirabassi, R. (2014). How to quality control check your RNA samples. Bitesizebio, 5(2), 1.