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.
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
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.
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