NEXTFLEX® Poly(A) Beads 2.0

The kit offers a convenient method for the purification of pure, intact mRNA upstream of RNA-Seq library prep. Poly(A)-tailed mRNA is isolated via magnetic beads conjugated to oligo(dT), and separation using a magnetic stand allows for high mRNA recovery. The intact mRNA is eluted in small volumes, thereby eliminating the need for precipitation. These beads have been validated with the new and improved NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0.

Features:

• 10 ng – 5 µg total starting RNA

• Magnetic bead-based protocol

• No organic solvents or precipitation step required

• Validated using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0

• Magnetic bead-based protocol for purification of pure, intact mRNA upstream of RNA-Seq library prep (no organic solvents or precipitation step required)

• Optimized for use with 10 ng – 5 µg of total RNA as starting material

• Validated using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0 and automated on the Sciclone® G3 NGSx and Zephyr® G3 NGS workstations

Kit Specs:

NEXTFLEX® Poly(A) Beads 2.0 for Reducing Ribosomal rRNA Reads in RNA-seq Libraries 

Several approaches have been used to remove ribosomal RNA (rRNA) from total RNA samples for RNA-Seq library preparation. Removal of rRNA avoids the waste of reagents and bioinformatics resources to sequence and align ~85% of total RNA comprising rRNA (which is generally not a target of interest in NGS sequencing experiments). PerkinElmer’s NEXTFLEX® Poly(A) beads 2.0 kit offers an easy and cost-effective method for removing rRNA in RNA-Seq libraries. This product takes advantage of the tract of adenosine residues present at the 3’ends of a vast majority of protein-coding mRNAs. Hybridization of these poly(A) tails to tracts of complementary thymidines (“oligo dT”) immobilized on solid supports allows the retrieval of poly(A)+ RNAs by recovering the solid support along with the hybridized RNA complexes.

Figure 1.Example of 1 µg of Universal Human Reference RNA (Agilent® # 740000) after Poly(A) enrichment using the NEXTFLEX® Poly(A) Beads 2.0.  3 µL Poly(A) enriched RNA was run on the LabChip® GXII Touch™ HT instrument using the RNA Pico Assay Reagent Kit (# CLS960012) and a DNA/RNA/Charge Variant Assay LabChip (# 760435)

Figure 2. NEXTFLEX® Poly(A) Beads 2.0 yield clean mRNA with minimal rRNA carry-over from 5 µg of total RNA input from (A) mouse brain and (B) MCF7 cells. ~1.5% rRNA carry-over was observed based on sequencing data.

NEXTFLEX® POLY(A) BEADS KIT 2.0 CONTENTS

• NEXTFLEX® Poly(A) Beads 2.0

• NEXTFLEX® Poly(A) Elution Buffer 2.0

• NEXTFLEX® Poly(A) Binding Buffer 2.0

• NEXTFLEX® Poly(A) Washing Buffer 2.0

REQUIRED MATERIALS NOT PROVIDED

• 10 ng -5 μg total RNA

• DynaMag™-2 Magnet (Life Technologies Cat # 123-21D) / or / similar

• Water bath / Heat blocks

• Tube Rotator-unit (VWR Cat # 13916-822) / or / similar

• 2, 10, 20, 200 and 1000 μL pipettes

• RNase-free pipette tips

• Nuclease-free test tubes and microcentrifuge tubes

• Microcentrifuge

• Vortex