rRNA Depletion

What is an rRNA depletion?

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!

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.

In this step, the RNA is measured using Thermo Fishers Qubit. 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.