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GeneRead rRNA Depletion Kits

For highly selective and efficient removal of rRNA and globin mRNA for NGS applications
  • Highly efficient removal of more than 99.9% of all types of ribosomal RNA
  • Unbiased retention of other RNA types
  • Effective rRNA depletion from human, mouse, and rat samples
  • Improved signal-to-noise ratio for sensitive detection of low-abundance RNAs
  • Optimal use of expensive sequencing technology

Ribosomal RNA (rRNA) comprises 85–90% of total RNA, taking up valuable sequencing capacity and resulting in a high signal-to-noise ratio that can make detection of the RNA species of interest difficult. GeneRead rRNA Depletion Kits effectively remove ribosomal RNA, while ensuring complete recovery of mRNA and noncoding RNA from various species, including human, mouse, and rat. By improving the ratio of useful data, decreasing bias, and preserving non-coding RNA types, the kits provide high-quality RNA that is especially suited for next-generation sequencing (NGS) applications. In addition to rRNA, globin mRNA can be effectively depleted from total RNA isolated from human whole blood with GeneRead Globin mRNA Depletion Probes. Procedure can be automated on the QIAcube.

Cat No./ID: 180211
GeneRead rRNA Depletion Kit (6)
$895.00
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For 6 x 100 µl reactions: HMR rRNA Depletion Probes, Antibody Solution, Hybridization Buffer, BioMag Protein G Beads, RNase-Free Water, RNase Inhibitor, Small Spin Columns, Reaction Tubes
Cat No./ID: 180224
GeneRead rRNA Depletion Nano Kit (48)
$4,574.00
Add To Cart
For 48 x 50 μl reactions: HMR rRNA Depletion Probes, Antibody Solution, Hybridization Buffer, BioMag Protein G Beads, RNase-Free Water, RNase Inhibitor, Small Spin Columns, Reaction Tubes, and the RNeasy MinElute Cleanup Kit
Cat No./ID: 180950
GeneRead Globin mRNA Depletion Probes
$174.00
Add To Cart
For 25–50 reactions; to be used with the GeneRead rRNA Depletion Kit (cat. no. 180211) or the GeneRead rRNA Depletion Nano Kit (cat. no. 180224)

GeneRead rRNA Depletion Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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More effective and specific rRNA depletion.

Total RNA from Jurkat cells was treated according to the manufacturer's instructions for each depletion or enrichment procedure. All depleted/enriched samples were made into libraries using the same method and sequenced in multiplex by Illumina sequencing on a MiSeq system. Sequencing reads were mapped by Bowtie 2 against the Ensembl RNA database and the percentage of mapped reads from each library for the Ensembl biotypes were plotted.

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Specific rRNA depletion for better representation of protein coding genes.

The abundance of specific proteincoding genes present in a sample following depletion of rRNA using kits from Suppler E, Supplier I, or the GeneRead rRNA Depletion Kit, was compared to a poly A purification method. Both the number of depleted genes and the magnitude of the depletion is significantly lower using the GeneRead rRNA Depletion Kit than with kits from Supplier E or Supplier I, which resulted in non-specific depletion of more than 70 times as many genes by a factor of 10 or more than the GeneRead rRNA Depletion Kit.

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Highly efficient rRNA removal.

A The lack of any rRNA peak in QIAxcel trace data demonstrates complete removal of rRNA in the rRNA-depleted sample, in contrast to the total RNA sample. B qRT-PCR assays indicate that more than 99% of all rRNA species, including mitochondrial rRNA, are removed with the kit
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GeneRead rRNA Depletion Kit procedure.

Specific oligonucleotides probes are hybridized to rRNA, bound to an antibody, and captured on a protein G bead. Removal of the beads effectively eliminates rRNA from the sample, which is then cleaned using the RNeasy MinElute Cleanup Kit.

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GeneRead combines two steps of specificity: DNA/RNA sequence specificity and antibody binding.

A When the oligonucleotide probes are perfectly matched, both direct capture (e.g., using biotin-streptavidin beads) and antibody-mediated capture (GeneRead rRNA Depletion Kit) will successfully capture the rRNA targets. B When probes cross-hybridize to RNA species other than rRNA, the mismatched probe/RNA will not be recognized by the GeneRead rRNA Depletion system, thereby avoiding non-specific depletion. In contrast, with direct capture, this second level of specificity does not exist and other RNA species may be co-depleted along with the rRNA.
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The GeneRead rRNA Depletion Kit ensures greater conservation of the mRNA profile.

The number of reads mapped to protein coding genes after sequencing of each depletion method was compared to those resulting from a poly A enrichment method to determine the specificity of the depletion method. (The purple dots indicate reads from mRNA species without poly A tails [e.g., histones] that are lost in poly A-based enrichment). The high linear correlation (R2) values of these results demonstrate that, compared to the rRNA depletion kits of Suppliers I and E, the GeneRead rRNA Depletion Kit (QIAGEN) is the best at preserving the profile of mRNA present in the original sample.

Performance
NGS research increasingly depends on the ability to obtain high-quality sequencing data from complex samples and limited starting materials. QIAGEN, with its leading expertise in sample preparation technologies, offers a portfolio of dedicated products for the next-generation sequencing workflow, including innovative sample enrichment solutions such as ribosmal RNA (rRNA) depletion. By eliminating the rRNA component from the total RNA sample, the GeneRead rRNA Depletion Kit makes it possible to achieve clear NGS results from complex samples.

Efficient removal of all rRNA species
The GeneRead rRNA Depletion Kit efficiently removes all types of rRNA, including the 4 main ribosomal RNAs, as well as mitochondrial rRNA. QIAxcel data showing rat spleen RNA demonstrates complete removal of the 18S and 28S ribosomal RNA peaks, compared to total RNA (see figure "Highly efficient rRNA removal"). When depleted samples are analyzed by quantitative RT-PCR, it is seen that more than 99.9% of all types of ribosomal RNA are removed with the kit.

Highly specific rRNA depletion
The biotype distribution of sequenced RNA following rRNA depletion demonstrates that the GeneRead rRNA Depletion Kit effectively eliminates rRNA, while preserving more miRNA and other noncoding RNA than rRNA depletion kits from other suppliers (see figure "More effective and specific rRNA depletion"). The biotype distribution of RNA prepared using kits from Suppliers E and I revealed significantly less effective rRNA elimination, while the kit from Supplier E enriched for scRNA (e.g., 7SL and Alu RNA). With the GeneRead rRNA Depletion Kit, the distribution of multiple RNA types, such as miRNA and other noncoding RNAs, is also preserved compared to poly A purification, which enriches for protein-coding RNAs.

No artificial depletion of protein-coding genes
The GeneRead rRNA Depletion Kit effectively depletes rRNA without affecting the natural representation of other RNA types. Kits from other suppliers may skew the representation of protein-coding (poly A) RNAs in a sample, particularly by nonspecific removal of non-ribosomal RNA. In contrast, the GeneRead rRNA Depletion Kit demonstrates greater concordance with poly A purification and preserves the natural representation of other RNA species and protein-coding genes (see figures "The GeneRead rRNA Depletion Kit ensures greater conservation of the mRNA profile" and "Specific rRNA depletion for better representation of protein coding genes").

Successful depletion of rRNA from 100 ng to 1 µg of human, mouse, or rat total RNA
The GeneRead rRNA Depletion Nano Kit efficiently removes >99.9% of all types of rRNA from as little as 100 ng total RNA, while ensuring complete recovery of mRNA and non-coding RNA from various species.
 
Effective depletion of globin mRNA together with rRNA
GeneRead Globin mRNA Depletion Probes effectively deplete globin mRNA in addition to rRNA from 100 ng to up to 5 µg of total RNA isolated from human whole blood and are to be used in combination with either the GeneRead rRNA Depletion Kit or the GeneRead rRNA Depletion Nano Kit.
Principle
Ribosomal RNA is the most abundant molecule in total RNA, making up over 80% of the total amount. In applications such as RNA sequencing, it is of great interest to maximize the amount of information received from a sequencing run. Ribosomal RNA provides little information about the transcriptome, and wastes valuable sequencing resources. The GeneRead rRNA Depletion Kit effectively removes ribosomal RNA (rRNA) while ensuring complete recovery of mRNA and noncoding RNA from a wide variety of species (see figure "GeneRead combines two steps of specificity: DNA/RNA sequence specificity and antibody binding"). In contrast to poly A enrichment, rRNA depletion preserves information on non-adenylated, non-coding, and regulatory RNAs, enabling investigation of RNA regulation, nascent transcription, RNA editing, and other phenomena that increase our understanding of the transcriptome's complexity. By improving the ratio of useful data, decreasing bias, and preserving non-coding RNA species, the kit provides high-quality RNA that is especially suited for NGS applications.
Procedure
The GeneRead rRNA Depletion Kit uses a highly efficient and specific hybridization method to selectively deplete rRNA (see figure "GeneRead combines two steps of specificity: DNA/RNA sequence specificity and antibody binding"). The method is based on hybridization of specific oligonucleotide probes that are designed to hybridize to the large (18s, 28s), small (5s, 5.8s), and mitochondrial (12s, 16s) rRNAs. The RNA:DNA hybrid is recognized by an antibody and the resulting antibody-hybrid complex is efficiently captured on a protein G bead. These beads can then be separated from the sample, removing the rRNA in the process (see figure "GeneRead rRNA Depletion Kit procedure"). The rRNA-depleted sample retains the diversity of RNA species, including poly A mRNA, non-adenylated mRNA, non-coding RNA, and regulatory RNAs.

Unlike affinity-tag approaches, only hybridized probes will be recognized by the antibody and bead capture system. This leads to very high efficiencies and fast reaction times. Residual probes are removed during RNA cleanup using the RNeasy MinElute Cleanup Kit. The majority of the procedure, from hybridization and capture through to RNA cleanup, is automatable on the QIAcube.
Applications

RNA prepared using the GeneRead rRNA Depletion Kits is suited for use in a wide range of downstream applications, such as:

  • Next-generation sequencing
  • Quantitative, real-time PCR
  • Microarray analysis

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