Product Class: Kit

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

Product Introduction

Globin mRNA and rRNA are highly abundant in blood samples, and their efficient and specific removal is desirable in order to reveal the biological significance of less abundant transcripts.

The NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) employs the NEBNext RNase H-based RNA depletion workflow to deplete globin mRNA, cytoplasmic rRNA and mitochondrial rRNA with human, mouse and rat samples, and is effective with intact and degraded RNA.

  • Efficient, specific depletion of rRNA and globin mRNA (adult, fetal and embryonic)
  • Suitable for low-quality or high-quality RNA
  • Compatible with a broad range of input amounts: 10 ng - 1 μg
  • Optional integration with poly(A) mRNA isolation workflows for removal of globin RNAs, rRNAs, and noncoding RNAs
  • Fast workflow: 2 hours, with less than 10 minutes hands-on time

The kit is also available with RNAClean® beads.

Catalog # Size Concentration
E7750S 6.0 reactions
E7750L 24.0 reactions
E7750X 96.0 reactions
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Product Information

Description

The great majority of RNA in blood samples is comprised of cytoplasmic and mitochondrial ribosomal RNAs (rRNA) and globin mRNA. These highly abundant RNA species can conceal the biological significance of less abundant transcripts, and so their efficient and specific removal is desirable.

The NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) employs the NEBNext RNase H-based RNA depletion workflow to deplete adult, embryonic and fetal globin mRNA (HBA1/2, HBB, HBD, HBM, HBG1/2, HBE1, HBQ1 and HBZ), cytoplasmic rRNA (5S, 5.8S, 18S, 28S, ITS and ETS) and mitochondrial rRNA (12S and 16S).

The kit is effective with human, mouse and rat total RNA preparations, both intact and degraded. The resulting depleted RNA is suitable for RNA-seq, random-primed cDNA synthesis, or other downstream RNA analysis.

This kit can also be used following poly(A) mRNA enrichment (e.g., using the NEBNext poly(A) mRNA Magnetic Isolation Module NEB #E7490), so that the final depleted RNA contains only mRNA of interest and no non-coding RNA. 
 
The kit is also available with RNAClean® beads.
 
Features

  • Efficient, specific depletion of rRNA and globin mRNA (adult, fetal and embryonic)
  • Suitable for low-quality or high-quality RNA
  • Compatible with a broad range of input amounts: 10 ng - 1 μg
  • Optional integration with poly(A) mRNA isolation workflows for removal of globin RNAs, rRNAs, and noncoding RNAs
  • Fast workflow: 2 hours, with less than 10 minutes hands-on time

For use with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) (NEB #E7416), refer to the Protocols tab for UMI Adaptors-specific guidance.

 
Figure 1. Depletion of globin mRNA and ribosomal RNA enriches for RNAs of interest across species



Human, mouse and rat whole blood total RNA (1 µg) was depleted of rRNA alone, or rRNA and globin mRNA transcripts, using the NEBNext Globin & rRNA Depletion Kit.  RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). Reads were identified as rRNA or globin mRNA using mirabait (6 or more, 25-mers), and levels of rRNA and globin mRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering.




Figure 2: Consistent Depletion Across Species and Across Inputs



Human, mouse and rat whole blood total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit or Globin-Zero® Gold rRNA Depletion Kit (Illumina®). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). Reads were identified as ribosomal or globin using mirabait (6 or more, 25-mers), and levels of rRNA and globin mRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates and error bars indicate standard error. The NEBNext Globin Depletion Kit is superior at depleting rRNA across species, and at depleting over 99% of globin mRNA.




Figure 3: Transcript expression correlation is maintained after depletion



Human whole blood total RNA (1 µg) was depleted of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit or Globin-Zero® Gold rRNA Depletion Kit (Illumina®). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). GENCODE v27 transcript abundances were estimated using Salmon. TPM (Transcript Per Million mapped reads) of protein coding transcripts, and R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates better transcript expression correlation between depleted and undepleted samples for the NEBNext Globin & rRNA Depletion Kit. Treatment does not alter the abundances of non-targeted transcripts.




Figure 4: Transcript expression correlation is maintained across a range of input amounts



Human whole blood total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit. RNA-seq libraries were prepared using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). GENCODE v27 transcript abundances were estimated using Salmon. TPM (Transcript Per Million mapped reads) and R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates consistent transcript expression regardless of input amount.




This product is related to the following categories:
RNA Depletion & mRNA Enrichment,
RNA Library Prep for Illumina,
This product can be used in the following applications:
RNA-seq,
RNA Analysis

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7750S     -20    
  • E7750L     -20    
  • E7750X     -20    

Properties & Usage

Materials Required but not Supplied

Magnetic rack/stand such as the NEBNext Magnetic Separation Rack (NEB #S1515)

80% Ethanol (freshly prepared)

Thermocycler

Thin wall 200 µl PCR tubes

Bioanalyzer®, TapeStation® (Agilent Technologies, Inc.) or similar fragment analyzer and consumables

Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) with NEBNext Ultra II RNA Library Prep (NEB #E7750 & #E7755)?
     

Manuals

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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What species can I use the NEBNext® Globin mRNA & rRNA Depletion Kit for? Will it work for other species?
  2. Which globin mRNAs and rRNAs are depleted with the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat)? 
  3. Can I use the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) with degraded RNA or fragmented RNA?
  4. What is the total RNA input I should use?
  5. Why must the RNA be free of DNA?
  6. What is the expected RNA yield recovered after globin mRNA and rRNA depletion?
  7. What is the percentage of globin mRNA and rRNA left after depletion?
  8. How can I determine if the globin mRNA and rRNA depletion was efficient?
  9. I am only interested in coding RNA. Can I use the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) to remove globin mRNA from previously enriched mRNA.
  10. Does the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications?
  11. Can I use Agencourt® AMPure® XP Magnetic Beads instead of NEBNext® RNA Sample Purification Beads (RNAClean® XP Magnetic Beads)?
  12. Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads?
  13. 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.