rRNAs are highly abundant in most cells, but despite being essential for all living organisms, they are in general of little interest for researchers using NGS. If they are present in the sequencing sample, a huge number of sequence reads will be wasted on unwanted molecules. So the cleanup of the rRNA is critical in RNA-Seq.
There are three ways of avoiding rRNA. Two of them – Poly(A) RNA selection and depletion of rRNA (ribo-depletion) are most popular, while the third one – target enrichment via size selection is less general due to its applicability mostly to the small ncRNA sequencing. Poly(A) selection enriches for the molecules of interest – the mRNAs, while the rRNA depletion eliminates the unwanted molecules – the rRNA. The choice of which of these methods should be used depends on what kind of RNA the researcher wants to see.
Since 2015 both of these commonly used rRNA removal options are available in the Lexogen portfolio and both kits, RiboCop for ribo-depletion, and the Poly(A) RNA Selection kit have been updated in 2020.
RiboCop uses magnetic bead-based depletion of rRNAs. The probes in RiboCop are carefully designed to selectively target only the rRNA, not influencing any other transcripts. They efficiently hybridize with diverse rRNAs and eliminate them from the total RNA fraction. It does not involve any enzymatic processes that reduce the reproducibility, keeping the protocol very simple, robust and straightforward with just 1.5 hours of prep time.
The foremost priority in the design of probes was not to distort the expression profile of the sample while keeping its depletion efficiency as high as possible, as we witnessed that this is the case with some kits. Also, we wanted to make it very cost-efficient, because in several cases the ribo-depletion was taking the biggest portion of cost in RNA-Seq sample preparation. We are proud to say that RiboCop successfully accomplished both of these objectives at satisfactory level.
It can be used together with any RNA-Seq library preps including CORALL Total RNA-Seq and other generic library preparation protocols from various manufacturers.
The Poly(A) RNA Selection kit was originally released in 2015 and was updated to V1.5 in 2020. This kit is also part of the CORALL mRNA-Seq bundle for whole transcriptome mRNA sequencing workflows. Poly(A) enrichment allows to focus the reads obtained in a sequencing experiment to protein-coding RNAs (95 % of the reads) by efficient removal of non-poly adenylated transcripts, such as cytoplasmic rRNAs.
Stringent Poly(A) selection is one of the most important steps in mRNA-Seq, and we conducted a very thorough investigation for finding the best beads in combination with the most ideal buffer composition.
With the addition of these two kits, we are very happy that we are providing a complete RNA-Seq pipeline, from extraction of RNA, enrichment and depletion of RNA molecules, sequencing library preparation, and the data analysis, thereby enabling complete transcriptome analysis, a slogan that we have been keeping in our mind all the time.
