Product Class: Kit

NEBNext® rRNA Depletion Kit (Human/Mouse/Rat)

An updated version of this kit (NEB #E7400) is available for superior performance.

Note that RNA Sample Purification beads are not included in this kit. If beads are required, we recommend ordering the NEBNext rRNA Depletion Kit v2 with Sample Purification Beads (E7405), but the NEBNext rRNA Depletion Kit with Sample Purification Beads (NEB #E6350) is also available.

Product Introduction

Ribosomal RNAs (rRNAs) are extremely abundant, constituting 80–90% of total RNA. Efficient removal of rRNA is critical to enable cost-effective sequencing of RNA samples, but this can be especially challenging with low quality RNA (e.g. FFPE RNA) and with low input amounts. The NEBNext rRNA Depletion kit employs the efficient RNase H method, as well as complete probe tiling of rRNA, thereby ensuring that even degraded rRNA is hybridized and subsequently removed.

  • Suitable for low-quality (e.g. FFPE) or high-quality RNA
  • Compatible with a broad range of input amounts: 5 ng - 1 μg
  • Fast workflow: 2 hours, with less than 10 minutes hands-on time
Catalog # Size Concentration
E6310S 6.0 reactions
E6310L 24.0 reactions
E6310X 96.0 reactions
Need a custom/large volume order? Contact Us

Product Information

Description



The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) employs an RNaseH-based method (1,2) to deplete both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations. This product is suitable for both intact and degraded RNA (e.g. FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-Seq, random-primed cDNA synthesis, or other downstream RNA analysis applications.

Now also available with optional Agencourt® RNAClean® XP beads for RNA purification.



Figure 1. Efficient Removal of rRNA from Intact and Degraded RNA (FFPE), While Retaining Coding and Non-coding Transcripts
Figure 1. Efficient Removal of rRNA from Intact and Degraded RNA (FFPE), While Retaining Coding and Non-coding Transcripts
RNA-seq libraries were generated from Universal Human Reference Total RNA (UHR, Agilent) or Breast Cancer FFPE RNA (with an archive age of 1 year and 10 years). RNA?was either untreated or treated with the NEBNext poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit. RNA-seq libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420). Reads were mapped to the hg19 genome and read distributions were determined using Picard RNA-seq Metrics. Libraries generated from rRNA-depleted RNA result in comparable low rRNA reads to poly(A) mRNA-enriched RNA, while also retaining more noncoding reads. rRNA depletion efficiency is achieved even with FFPE RNA.


Figure 2. NEBNext rRNA Depletion Kit Workflow Figure 2. NEBNext rRNA Depletion Kit Workflow
Figure 3. NEBNext rRNA Depletion Kit Workflow TimesFigure 3. NEBNext rRNA Depletion Kit Workflow Times
Figure 4. Depletion Efficiency with Intact or Degraded RNA
Figure 4. Depletion Efficiency with Intact or Degraded RNA
Ribosomal RNA was depleted from intact Universal Human Reference Total RNA (UHR, Agilent) (RIN>9) and degraded UHR Total RNA (RIN<3) using either the NEBNext rRNA Depletion Kit, Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre) or Ribo-Zero Gold provided within the TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina). rRNA- depleted RNA libraries were made using either the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420. Green and blue bars) or the TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina, red bars). Total rRNA-aligned reads were determined using Bowtie 2.0 (local, sensitive). NEBNext rRNA-depleted libraries contain a minimal percentage of rRNA reads regardless of the quality of the RNA.
Figure 5. Depletion Efficiency with FFPE RNAFigure 5. Depletion Efficiency with FFPE RNA

0.1 μg, 0.5 μg or 1 μg of breast cancer FFPE RNA samples (with archive ages of one year and 10 years) were depleted of rRNA using either the NEBNext rRNA Depletion Kit or the TruSeq® Stranded Total RNA Kit with Ribo- Zero Gold (Illumina #RS-122-2301). rRNA-depleted RNA libraries were made using either the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420)(green bars) or the TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (blue bars). Total rRNA-aligned reads were determined using Bowtie 2.0 (local, sensitive). NEBNext rRNA-depleted FFPE libraries result in a minimal percentage of rRNA reads, regardless of the archive age of the FFPE RNA.
Figure 6. rRNA Depletion Efficiency on Mouse and Rat RNAFigure 6. rRNA Depletion Efficiency on Mouse and Rat RNA

Ribosomal RNA was depleted from mouse and rat kidney total RNA (two technical replicates) using the NEBNext rRNA Depletion Kit. RNA-seq libraries were made from non-depleted Total RNA or rRNA-depleted RNA, using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420). Total rRNA-aligned reads were determined using Bowtie 2.0 (local, sensitive). NEBNext rRNA-depleted mouse and rat libraries contain a minimal percentage of reads mapping to rRNA.
Figure 7. Transcript Expression Correlation with Non-depleted LibrariesFigure 7. Transcript Expression Correlation with Non-depleted Libraries

Libraries were made from UHR RNA (Agilent) and Breast Cancer FFPE RNA (with archive age of one year and 10 years), both non-depleted and depleted rRNA using the NEBNext rRNA Depletion Kit. All libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420). TopHat2 and Cufflinks were used for read mapping and transcript assembly and quantification. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) correlation analysis indicates very good transcript expression correlation (R >0.93) between Depleted and Non-Depleted libraries. NEBNext rRNA depletion does not affect transcript expression levels.


Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E6310S     -20    
  • E6310L     -20    
  • E6310X     -20    

Properties & Usage

Materials Required but not Supplied

  • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
  • Thermal Cycler
  • Agencourt® RNAClean® XP (Beckman Coulter, Inc., Cat. #A63987)

References

  1. Adiconis, X. et al (2013). Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nature Methods 10. 623-629.
  2. Morlan,J.D. et al. (2012). Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One 77. e42882.

Protocols, Manuals & Usage

Protocols

  1. Protocol for use with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310, E6350)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

FAQs & Troubleshooting

FAQs

  1. What is the expected RNA yield recovered after rRNA depletion?
  2. What is the total RNA input I should use?
  3. What is the percentage of rRNA left after depletion?
  4. Can I use purification methods other than Agencourt RNAClean XP Magnetic Beads?
  5. Can I use Agencourt AMPure XP Magnetic Beads instead of RNAClean XP Magnetic Beads?
  6. Can I use this product with degraded RNA or fragmented RNA?
  7. To remove ribosomal RNA from total RNA, should I use the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit (NEB #E6310)?
  8. Does the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) work for ribosomal footprinting or profiling application?
  9. Does the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications?
  10. What total RNA input should I use?
  11. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

This product and its use are subject to one or more issued and/or pending U.S. and foreign patent applications. Commercial use for the determination, prediction and prognosis of cancer, or response to therapy for cancer, in humans or animals may require a license. Please contact busdev@neb.com for more information.