Product Class: Kit

NEBNext® High Input Poly(A) mRNA Isolation Module


Catalog #E3370

Product Introduction

The NEBNext High-Input Poly(A) mRNA Magnetic Isolation Module is designed to isolate intact poly(A)+ RNA from high inputs (5-50 µg per 0.2 ml reaction) of previously isolated total RNA, using oligo d(T)25-coupled paramagnetic beads. Intact mRNA can be obtained in approximately one hour, and eluted in small volumes.

  • High inputs: 5 – 50 µg total RNA (DNA-free)
  • Low elution volume
  • Fast workflow
  • Automation-compatible

Product Information

Description

The NEBNext High-Input Poly(A) mRNA Magnetic Isolation Module isolates intact poly(A)+ RNA from high inputs (5-50 µg per 0.2 ml reaction) of previously isolated total RNA. The technology is based on the coupling of oligo d(T)25 to paramagnetic beads, which is then used as the solid support for the direct binding of poly(A)+ RNA. The procedure permits the manual processing of multiple samples and can be adapted for automated high-throughput applications. Additionally, magnetic separation technology permits elution of intact mRNA in small volumes, thereby eliminating the need for precipitation of the poly(A)+ transcripts in the eluent. Intact poly(A)+ RNA, which is representative of the mRNA population of the original sample, can be obtained in approximately one hour.

For inputs less than 5 µg, the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) is recommended.

Figure 1:  The NEBNext High Input Poly(A) mRNA Isolation Module provides consistent representation across a wide input range

E3370 Wide Range

A. Poly(A) RNA enriched using the NEBNext High Input Poly(A) mRNA Isolation module from 50 μg or 5 μg of total Universal Human Reference (UHR) RNA (Agilent®) containing External RNA Controls Consortium (ERCC) RNA and Spike-In RNA Variants (SIRV Set 4, Lexogen®). Total yield was assessed using Qubit® RNA High Sensitivity Assay Kit (Invitrogen®). Poly(A) RNA is typically ~1-5% of total RNA. 
B. Bioanalyzer® (Agilent) traces for Total UHR RNA and poly(A)-enriched RNA from 50 μg or 5 μg total RNA input. 
C. Transcript abundances were determined from libraries prepared from 40 ng (50 μg input) or 8 ng (5 μg input) poly(A)-enriched RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit and sequenced on an Illumina® NextSeq® 550 sequencer. Six million reads were sampled from each library.


 
 
Figure 2: The NEBNext High Input Poly(A) mRNA Isolation Module produces low ribosomal RNA retention, across sample types

E3370 Retention

Poly(A) RNA was enriched using Dynabeads® mRNA Purification Kit (Invitrogen), Poly(A)Purist™ MAG (Invitrogen) or the NEBNext High Input Poly(A) mRNA Magnetic Isolation Module from 50 μg Universal Human Reference RNA (UHR, Agilent) or RNA extracted from mouse kidney tissue or S. cerevisiae (yeast) using the Monarch® Total RNA Miniprep Kit. 
A. Representative Bioanalyzer traces of total and poly(A)-enriched RNA. 
B. Percent ribosomal RNA (rRNA) of total or poly(A)-enriched RNA samples was determined from sequencing of triplicate (UHR and mouse poly(A) samples) or duplicate (total RNA and yeast poly(A) RNA samples) experiments, with standard deviation. Libraries were prepared from 40 ng poly(A)-enriched RNA using the NEBNext Ultra™ II Directional RNA Library Prep Kit and sequenced on an Illumina NextSeq 550 instrument. Six million reads were sampled from each library.


 
 
Figure 3: RNA from the NEBNext High Input Poly(A) mRNA Isolation Module produces higher library yields for nanopore sequencing, with good read mapping

E3370 Higher Yield 
400 ng of poly(A)-enriched Universal Human Reference (UHR) RNA, enriched using the stated methods, was prepared for Direct RNA Sequencing (ONT #SQK-RNA002) on a GridION® sequencer (Oxford Nanopore Technologies®). 100 ng of poly(A)-enriched UHR RNA, enriched using the stated methods, was prepared for Direct cDNA Sequencing (ONT #SQK-DCS109) on a GridION sequencer. 
A. Library yields were assessed using Qubit dsDNA High Sensitivity Assay Kit (Invitrogen); shown are the average of replicates with standard deviation. 
B. Average mapping percentages of reads from replicate Direct RNA and Direct cDNA sequencing runs with standard deviation.


 
 
Figure 4: The NEBNext High-Input Poly(A) mRNA Isolation Module yields high-quality poly(A) mRNA for direct RNA and cDNA Sequencing

E3370 High Quality

5´➝3´ transcript coverage of the top one thousand transcripts from representative Direct RNA (A) and Direct cDNA (B) sequencing runs on the Oxford Nanopore Technologies platform with libraries prepared from poly(A)-enriched UHR RNA using the NEBNext High Input Poly(A) mRNA Isolation Module.


 
This product is related to the following categories:

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E3370S     4    
      NEBNext RNA Binding Buffer (2X) E7492AVIAL 4 1 x 7.2 ml Not Applicable
      NEBNext Wash Buffer E7493AVIAL 4 1 x 28.8 ml Not Applicable
      Nuclease-free Water E7495AVIAL 4 1 x 1.2 ml Not Applicable
      NEBNext Tris Buffer E7496AVIAL 4 1 x 6 ml Not Applicable
      NEBNext® High Input Oligo d(T)25 Beads E3371AVIAL 4 1 x 0.48 ml Not Applicable

Properties & Usage

Features

  • High inputs: 5 – 50 µg total RNA (DNA-free)
  • Low elution volume
  • Fast workflow
  • Automation-compatible

Protocols, Manuals & Usage

Protocols

  1. Where can I find guidelines and protocols for using the NEBNext High Input Poly A mRNA Isolation Module (NEB #E3370)?

Manuals

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

FAQs & Troubleshooting

FAQs

  1. Can I isolate poly(A) mRNA directly from tissue lysis, using the NEBNext® High Input Poly(A) mRNA Isolation Module?
  2. Do I need to perform two rounds of binding?
  3. Can I use the NEBNext® High Input Poly(A) mRNA Isolation Module with total RNA inputs higher than 50 µg? What about lower inputs?
  4. Can the RNA be eluted with water?
  5. Can the NEBNext® High Input Oligo d(T)25 Beads be reused?

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.

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