RNA Depletion & mRNA Enrichment

RNA sequencing is a powerful technique for the analysis of cellular states, but since transcripts from abundant RNA species (e.g., ribosomal RNA (rRNA), globin messenger RNA (mRNA)) often comprise 80-90% of total RNA, this can overwhelm subtler biological phenomena, effectively drowning it out beneath a sea of noise. This leaves two options: 

  • mRNA enrichment – Selectively enriching for poly(A)-tailed transcripts
  • RNA depletion – Selectively depleting abundant/off-target transcripts 


mRNA Enrichment: 

mRNA enrichment is a commonly used method to enrich poly(A)-tailed mRNA from a sample of total RNA. It is most frequently achieved using oligo(dT) beads that bind to the poly(A) tail of mRNAs. Following a wash step, the mRNA is eluted from the beads and becomes the predominant species in the sample. This simple workflow is optimal for high quality RNA and poly(A)-tailed RNAs, which primarily includes proteins in eukaryotic cells that are intended for translation.  
The NEBNext® Poly(A) mRNA Magnetic Isolation Module enables the removal of rRNA and other non-poly(A)-tailed RNAs using Oligo d(T) beads, effectively enriching for poly(A)-tailed RNAs in a cost-effective manner. An Express protocol option is now available in the product manual, providing substantial time savings with a more streamlined workflow.


RNA Depletion: 

RNA depletion broadly describes the removal of unwanted, abundant RNA species from a sample. As rRNAs are the most abundant RNA species in most samples, RNA depletion often refers to rRNA depletion, but other species may also be targeted if they obscure a biologically significant readout, as in the case of the depletion of globin mRNA from blood samples. The NEBNext RNA depletion workflow utilizes targeted DNA probes and RNase H to selectively bind to and degrade abundant RNAs.  
Several NEBNext kits are predesigned for the depletion of certain RNA species from human, mouse, and rat samples: 

  • NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) – available with or without RNA sample purification beads
  • NEBNext rRNA Depletion Kit (Bacteria) – available with or without RNA sample purification beads 
  • NEBNext rRNA rRNA & Globin mRNA Depletion Kit (Human/Mouse/Rat) – available with or without RNA sample purification beads 

For more targeted depletion of known RNAs from samples of high or low quality, the NEBNext Custom RNA Depletion Design Tool can be used in tandem with the NEBNext RNA Depletion Core Reagent Set to design a set of closely tiled probes (to be ordered from your trusted oligo provider) and to deplete your unwanted RNAs from your sample, regardless of species.  


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Application Notes for RNA Depletion & mRNA Enrichment
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