rRNA stands for ribosomal RNA and constitutes a large part of the cells ribosomes. In fact, about 90% of the total RNA content of the cell is rRNA, with the rest being microRNA, tRNA and mRNA. While the rRNA has been very useful for RNA quality control in the previous step of the pipeline, it actually holds no genetic information of value for us and can be removed to make the RNA sample clearer and lighter in preparation for the coming steps.

Experiment

We performed our rRNA depletions using Thermo Fishers MICROBexpress Bacterial mRNA Enrichment Kit. This kit utilizes magnetic beads which are primed to capture and bind the rRNA to them. These beads are added to the sample. Using a magnet, the beads can then be pulled to the side of the sample tube and the eluate can be pipetted, giving us an RNA sample free of rRNA[1]!

As with more of the following steps of this pipeline, this procedure requires several chemicals and other reagents to function. These agents remain in the RNA sample after the depletion and can interfere with following steps of refining the RNA. They are removed by precipitation, which forces the RNA out of the solution and allows us to collect it by centrifugation[1, 2].

In this step, the RNA is measured using Thermo Fishers Qubit [3]. As with all work involving RNA, good sterile technique is required. us throughout the project.

Result

The purified RNA samples are checked for their concentrations after the precipitation to determine successful rRNA depletion. We use Qubit for this, as it is more precise and we have found that NanoDrop is inaccurate at the smaller concentrations introduced in this step.

Table 1: This table shows the nucleic acid concentrations of our four samples after rRNA depletion and precipitation. Two of them are worm samples (w16_1, w16_2), and two of them are controls (c16_1, c16_2). The concentrations and the subsequent total mRNA are good, with the exception of w16_1 which is below average.

Conclusion

These results can generally be seen as acceptable (a total nucleic acid content of 1000 ng is seen as the threshold) and can be moved on to the next step of the process.

In many cases, a successful rRNA depletion results in a loss of up to 90% of the total nucleic acid contents of the cell [4]. A significantly smaller loss may raise suspicions of inadequate rRNA removal. This may be due to several reasons - such as poor dispersion of the magnetic beads throughout the sample, causing less of the beads to bind the the rRNA molecules. It can also be due to poor separation of the magnetic beads from the eluate (eg. not enough time on the magnet), or by overloading the sample by introducing too much input RNA [1]. A gel electrophoresis of the finished rRNA depletion product can generally discern this - no rRNA bands at 1.5kb and 2.9kb on the gel along with a reasonable nucleic acid concentration (around 10-20% of input RNA) are results that we generally go forward with.

Precipitation

The EtOH precipitation is a necessary step in the purification of the RNA, where you want to separate the RNA from the liquid it is solved in. In most cases the RNA is solved in water where both the water molecules and RNA are charged and thereby interacts with each other, thus the RNA is hydrophilic. This is what we want to change. For us to separate the RNA from the water molecules we need to make the RNA less hydrophilic and make the pellet visible, which is done by adding the following.

Salt: The choice of salt varies between different situations, but we chose to use sodium acetate. The salt neutralizes the charges on the RNA, which makes the RNA less hydrophilic.

Ethanol: The ethanol on the other hand has the function of simplify the interaction between the nucleic acids and the salt. The salt in combination with the ethanol forces the nucleic acids to precipitate and can thereby be separated from the water with centrifugation.

Glycogen: It is important that the visibility of the RNA pellet is good enough to avoid touching it when pipetting the supernatant. When adding the glycogen the pellet gets more visible due to the fact that glycogen is a polysaccharide and cannot be solved in alcohols. Thereby when the glycogen is added into the RNA sample, nucleic acids will get trapped and get precipitated with the glycogen.

Glycoblue: Another complementary method to visualize the RNA pellet is to dye it. GlycoBlue is a blue dye that binds specific to glycogen, contributing of making the pellet even more visible. Thereby it will be easier for the user to pipette with less chance of touching the pellet.

References

[1] - Thermo Fisher, 2018. MICROBExpress™ Kit Protocol (PN 1905 Rev C) https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_057051.pdf Date of visit 2018-10-15

[2] - Walker, SE, Lorsch J. 2013. Chapter Nineteen - RNA Purification - Precipitation Methods. Methods in Enzymology, 530. p.337-343.

[3] - Thermo Fisher, 2018. Qubit Flourometric Quantitation. https://www.thermofisher.com/se/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit.html Date of visit 2018-10-15

[4] Petrova OE, Garcia-Alcalde F, Zampaloni C, Sauer K. 2017. Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes. Scientific Reports 7, Article number: 41114.