RNA sequencing is a genomic approach with a wide range of options. It has enabled many innovations and new therapeutic options in recent years. However, not all RNAs are the same. What are the main differences between the molecules? What is relevant to your project, and what do you need to consider? We want to explain these questions in more detail.
Before you start an RNA sequencing project, it is necessary to consider the differences in RNA molecules. Let’s start with the three main types, which are differentiated due to their function. These main types are called messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). The function of mRNA is to provide the template for protein synthesis. It is also called coding RNA. Transfer RNAs (tRNAs) carry amino acids to the ribosomes for protein biosynthesis. Ribosomal RNAs play a structural and catalytic role during translation.
However, not all RNA molecules fall into these three main categories. In addition to functional differentiation, RNA molecules are categorized according to their size. A distinction is made between molecules smaller than 200 nucleotides and larger than 200 nucleotides. Consequently, there are so-called small non-coding RNAs (sRNA) and long non-coding RNAs (lncRNA). Both types are not translated into protein but are widely expressed and have critical roles in gene regulation. Two known small non-coding RNA classes are microRNAs (miRNAs) and small interfering RNAs (siRNAs). Both are involved in RNA interferences (RNAi) to silence genes and regulate gene expression.
We can start a sequencing project with this knowledge of the different RNA types. Please have a look at figure 1. The RNA is isolated from the starting material in the first step, and a first selection is made. This first choice is necessary as not all isolation kits work well for all types of RNA molecules. If you are interested in small RNAs, you need to consider that with most kits, small RNAs (smaller than 100 nucleotides) cannot be isolated adequately; therefore, special kits need to be used. Thus, please always double-check if your preferred kit selects the RNA type you want to analyze. After successful isolation, the given workflow is straightforward for small RNA sequencing. With an appropriate library preparation kit, RNA is prepared for sequencing.
For larger RNA molecules, you can choose between two applications after isolation. The most common approach is coding RNA sequencing (mRNA sequencing). For this application, the mRNA is purified from the diversity of RNA molecules. This purification is done with poly-T oligonucleotides, which are attached to magnetic beads. The poly-A-tailed mRNAs bind to the poly-T oligonucleotides on the magnetic beads. After extracting the mRNAs, the sequencing library can be prepared exclusively based on the mRNA molecules. Since most eukaryotes have poly-A-tailed mRNAs, this RNA approach can be used to analyze eukaryotic cells. In contrast, the mRNA sequencing approach is unsuitable for bacterial samples because they lack the poly A-tail.
But how does the whole RNA sequencing (total RNA sequencing) approach compare? This approach captures mRNAs and long non-coding RNAs with a size larger than 100 nucleotides. After isolation, rRNA molecules have the largest share of the isolated RNAs. However, their information content is often not relevant to most research questions. This is why depleting rRNA is a recommended pre-treatment step before library preparation and sequencing.
Figure 1 | Differences in RNAs. Comparison of the target RNA molecules and the main steps of the different RNA sequencing approaches.
In summary, the different functions and sizes of RNA molecules must be considered before sequencing. Which RNA types you are interested in influences the choice of a suitable RNA isolation kit and library preparation steps – because not all RNAs are the same! For this reason, we offer Small RNA Sequencing, Coding Transcriptome Sequencing, and Whole Transcriptome Sequencing. We are happy to help you make the right selection.