rRNA Depletion vs Poly(A) Selection: Which Should You Use for RNA-Seq?
Team Dynamic Matrices | 5 October 2026
Every RNA-seq workflow has to deal with ribosomal RNA, which makes up roughly 80 to 90 percent of total RNA in a typical cell. There are two ways to stop it eating your reads: select the RNA you want (poly(A) selection) or remove the RNA you do not want (rRNA depletion). The right choice depends on your RNA, your organism and your question.
We make an rRNA depletion kit, so weigh what follows accordingly. We say plainly where poly(A) selection is the better choice.
The short answer
- Intact RNA from a eukaryote, and you need protein-coding gene expression: poly(A) selection. It is simple, inexpensive and gives the highest exonic coverage per read.
- Degraded or FFPE RNA, non-polyadenylated transcripts, bacteria, or an unknown mix: rRNA depletion.
- Not sure how good your RNA is: measure RIN or DV200 first, then choose.
How each method works
Poly(A) selection captures polyadenylated transcripts with oligo-dT, usually on magnetic beads, and discards everything else. rRNA has no poly(A) tail, so it is removed indirectly: this is an enrichment of mRNA rather than a depletion of rRNA. rRNA depletion works on the unwanted RNA directly, using probe hybridisation with bead pulldown, RNase H digestion or sequence-selective phase separation, and it keeps all other RNA whether or not it carries a tail. Our method comparison covers the main depletion approaches.
Side by side
| Poly(A) selection | rRNA depletion | |
|---|---|---|
| What is kept | Polyadenylated RNA, mainly mRNA | All RNA except the targeted rRNA |
| Non-polyadenylated RNA (histone mRNAs, many lncRNAs, circRNAs, most bacterial transcripts) | Lost | Retained |
| Degraded or FFPE RNA | Poor: the tail is separated from the transcript body, giving 3' bias and low yield | Usable: does not depend on an intact tail |
| Exonic coverage per read | Higher. In one comparison, depletion needed 20 to 50 percent more reads for the same exonic coverage (Zhao et al., 2018) | Lower |
| Transcript diversity | Narrower | Wider: more unique transcriptome features detected (Zhao et al., 2018) |
| Bacteria | Not suitable | Standard approach, with a probe panel or catcher library for the organism |
| Typical use | Gene expression from high-quality eukaryotic RNA | Whole transcriptome, archival and clinical samples, non-coding RNA |
When poly(A) selection is the right call
If your RNA is intact, the organism is a eukaryote and the question is protein-coding gene expression, poly(A) selection is hard to beat. In a comparison on human blood and colon RNA, polyA+ selection gave higher exonic coverage and more accurate gene quantification than rRNA depletion, and the depletion libraries needed 20 percent (blood) to 50 percent (colon) more reads to reach the same exonic coverage (Zhao et al., Scientific Reports, 2018). That is a real cost, not a footnote.
When rRNA depletion is the right call
- Degraded or FFPE RNA. Fragmentation separates the poly(A) tail from the rest of the transcript, so poly(A) selection recovers a small, 3'-biased fraction. See rRNA depletion for degraded RNA and FFPE RNA extraction challenges.
- Non-polyadenylated RNA matters. Histone mRNAs, many lncRNAs, circRNAs and nascent transcripts are lost with poly(A) selection.
- Bacteria and mixed communities. Poly(A) selection is not suitable. Depletion needs a probe panel or catcher library that covers the organism's rRNA.
- Discovery work. Depletion keeps the whole transcriptome, so you can find what you did not plan to look for.
What depletion costs you
Two things. First, you will typically sequence deeper for the same exonic coverage, as above. Second, depletion is never complete, and rRNA starts at 80 to 90 percent of the sample, so a method that removes 97 percent of it can still leave roughly 10 to 20 percent of reads on rRNA. Check what that costs in your budget with our worked cost model, and verify any depletion by qPCR for 18S or 28S before you sequence.
A quick decision guide
| Your situation | Choose |
|---|---|
| Intact RNA, eukaryote, protein-coding gene expression | Poly(A) selection |
| Intact RNA, eukaryote, lncRNA or other non-polyadenylated RNA | rRNA depletion |
| FFPE or degraded RNA with DV200 above about 30 percent | rRNA depletion |
| Very degraded RNA, DV200 below about 30 percent | Targeted capture of predefined genes, or do not sequence |
| Bacterial or mixed samples | rRNA depletion with an organism-matched panel |
| RNA quality unknown | Measure RIN or DV200 first (see RNA QC beyond A260/A280) |
References
- Zhao S, Zhang Y, Gamini R, Zhang B, von Schack D. Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion. Sci Rep 8, 4781 (2018).
- Adiconis X et al. Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Methods 10, 623–629 (2013).
- Schuierer S et al. A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples. BMC Genomics 18 (2017), doi:10.1186/s12864-017-3827-y.
- Herbert ZT et al. Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction. BMC Genomics 19, 199 (2018).
FAQs
Neither is better in general. Poly(A) selection gives higher exonic coverage per read on intact eukaryotic RNA; rRNA depletion is the better choice for degraded or FFPE RNA, non-polyadenylated transcripts and bacteria.
No, they are alternative strategies for the same underlying problem, not complementary steps. Combining both is uncommon and rarely necessary, and each extra step loses material.
Poly(A) selection captures the tail. When RNA is fragmented, only the pieces that still carry the tail are captured, and those come from the 3' end of the transcript, so coverage piles up there.
Yes. Depletion keeps mRNA along with all other non-rRNA RNA, so protein-coding expression can be quantified. Expect to sequence deeper than with poly(A) selection to reach the same exonic coverage.