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Ribogone Mammalian
Ribosomal RNA Removal Kit
Features:
• Effective rRNA removal method for human, mouse, and rat total RNA samples—depletes ≥95% of ribosomal RNA sequences
• Suitable for any quality RNA, including FFPE samples
• Low input working range: 10–100 ng starting material
• Works seamlessly with downstream random-primed cDNA synthesis kits like the Stranded RNA-Seq Kit or the SMARTer Universal Low Input RNA Library Prep Kit
Cat. #
Product
634846
Ribogone
TM
- Mammalian
634847
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more information
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24 Rxns
$3,649 + $264.9 GST
$3,101.65 + $310.17 GST
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Quote: 15offRibogone. Offer valid till the end of May, 2015
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rRNA removal from small quantities of RNA
The RiboGone-Mammalian kit specificallt removes ribosomal RNA (rRNA) and mitochondrial RNA (mtRNA) sequences from human, mouse or rat total RNA samples. The RiboGone technology is based on hybridization and
RNase H digestion that specifically depletes 5S, 5.8S, 18S, and 28S nuclear rRNA sequences as well as 12S mtRNA
sequences. The kit is desiged to work with any quality starting RNA, including degraded material like FFPE samples. The RiboGone-Mammalian kit is highly suited for sample preparations prior to random-primed cDNA synthesis and
RNA-seq or transcriptome analysis applications. Following the use of RiboGone-Mammalian, and the SMARTer
Stranded RNA-Seq Kit or the SMARTer Universal Low Input RNA Library Prep Kit, rRNA reads have been recorded
to be between 1-5% of total reads and lower.
RiboGone-Mammalian
• Input compatibility range: 10 ng to 100 ng
• Sample type: Human, Mouse and Rat
• Targets nuclear, and some mitochondrial,
ribosomal RNA
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more information
Click here to contact
Scientifix and receive 15% off
Quote: 15offRibogone. Offer valid till the end of May, 2015
Freight charges apply. Prices subject to change without notice.
Please contact Scientifix for details. Prices subject to change without notice.
1800 007 900
[email protected]
www.scientifix.com.au
c D N A
Begin with
total RNA
of any quantity
S Y N T H E S I S
Incubate
with RiboGene
Hyb Buffer
Add provided
enzymes and
buffer, and
incubate
&
L I B R A R Y
Purify
rRNA-depleted
RNA with
SPRI beads
C O N S T R U C T I O N
Synthesize
first-stranded
cDNA
using N6 priming
and SMART™
technology
~1 hr
Purify
first-strand
cDNA
1 hr, 45 min
PCR-amplify
RNA-Seq
library
Purify
RNA-Seq
library
Validate
RNA-Seq
library
20 min
45–60 min
50 min
Figure 1. Workflow for the SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian. This kit is designed for use with low-input
samples containing 10–100 ng of low-input total RNA samples. cDNA libraries generated using this kit are ready for Illumina sequencing and have ≥99%
accuracy in identifying strand of origin information.
Table I: Sequence Alignment Metrics
Human Universal Reference RNA (HURR)
Human Brain Reference RNA (HBRR)
6,829,540
7,728,850
No. of reads
Mapped to rRNA
Mapped to mitochondrial RNA
62,792
(0.9%)
49,844
(0.7%)
318,006
(4.7%)
224,939
(2.9%)
Mapped to RefSeq
4,871,900
(76%)
5,515,264
(75%)
Mapped uniquely to RefSeq
4,435,123
(70%)
4,888,340
(66%)
Exons
2,311,575
(47%)
2,712,444
(49%)
Introns
2,560,325
(53%)
2,802,820
Genes identified
14,563
(51%)
13,839
Table I. Sequencing alignment metrics for HURR and HBRR libraries. 10 ng samples of intact HURR and HBRR were used as input for the
SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian. Libraries were sequenced on an Illumina MiSeq platform with ~7M 1 x 50
bp single end reads per library. Reads were trimmed by CLC Genomics Workbench and mapped to rRNA and the mitochondrial genome with CLC (%
reads indicated). The unmapped reads were subsequently mapped with CLC to the human genome with RefSeq masking, producing mapped reads and
uniquely mapped reads. The number of genes identified in each library was determined by the number of genes with an RPKM (Read per Kilobase of
Exon per Million Reads) of at least 0.1. The number of reads that map to introns or exons is a percentage of the reads successfully mapped to RefSeq.
The RiboGone - Mammalian kit efficiently removes rRNA sequences from total RNA
The RiboGone - Mammalian kit can remove rRNA sequences from both intact and degraded RNA samples. The low
number of sequencing reads that map to rRNA is similar for RiboGone-treated and oligo(dT)-purified RNA samples.
Both intact (Human Brain Total RNA) and degraded (FFPE tissue) RNA samples are suitable for SMARTer Stranded
RNA-Seq Kits (Figure 2). The RiboGone method is based on selective hybridization to rRNA, leaving both mRNA and
non-coding RNAs available as templates for the reverse transcription reaction. Alternative methods for decreasing
the number of rRNA reads (such as oligo(dT)-based methods) also decrease the number of sequencing reads mapping to non-coding RNAs.
Distribution of Reads in Libraries
0.9%
1.1%
1.5%
100
12.7%
14.2%
16.2%
Introns
Exons
11.6%
75
65.8%
41.2%
Percent of Reads
Category
rRNA
Intergenic
87.1%
61.9%
50
Figure 2. Efficient rRNA removal with the RiboGone Mammalian kit. RNA-seq libraries were generated from Human
Brain Total RNA (Cat. No. 636530) or Breast Cancer FFPE RNA
(Cureline) extracted using a NucleoSpin totalRNA FFPE kit
(Cat. No. 740982.10) and treated with the indicated rRNA removal
method. Reads were mapped to the hg19 genome and read
distributions were determined using Picard RNA-Seq Metrics.
Libraries generated from RiboGone-treated RNA had comparably
low rRNA reads to oligo(dT)-enriched RNA (Cat. No. 9186) while
retaining more non-coding reads.
74.8%
5.6%
25
43.5%
19.7%
1.9%
5.7%
0
20.5%
9%
5.4%
Human
Brain
Untreated
Human
Brain
dT Purified
Human
Brain
RiboGone
Library
Breast Cancer
FFPE
Untreated
Breast Cancer
FFPE
RiboGone
2