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Cancer biomarker discovery

Cancer cell image

Cancer is a multigene disorder that arises from gene mutations as well as changes in transcriptional, epigenetic, and proteomic profiles. These changes can serve as valuable biomarkers for both early detection/diagnosis and the development of individualized therapy. Mutations in several known oncogenes (e.g., EGFR, HER2, KRAS) and tumor suppressor genes (e.g., TP53, PTEN, PI3K) are already being used as biomarkers to guide therapies in breast, ovarian, lung, and prostate cancers, among others.

Next-Generation Sequencing (NGS) analysis has greatly facilitated cancer biomarker discovery of circulating nucleic acids within blood, urine, saliva, pleural effusions, and cerebrospinal fluid (i.e., noninvasive liquid biopsies), which can contain tumor-derived genetic information (Siravenga et al. 2017). The sequencing of entire genomes or thousands of mutations simultaneously in a cost-effective manner with NGS can serve as a powerful tool in biomarker detection and discovery. The molecular profiles gathered from circulating tumor DNA (ctDNA) can be further complemented with those obtained through analysis of circulating tumor cells (CTCs), as well as RNA, proteins, and lipids contained within cell-derived vesicles, such as exosomes.

Recent advances in cancer biology have highlighted the importance of exosomes as carriers of genetic and biological messages between cancer cells and their immediate and distant environments. Cancer cells secrete exosomes containing diverse molecules that can be transferred to recipient cells and vice versa to induce a plethora of biological processes, including angiogenesis, metastasis formation, and therapeutic resistance. Therefore, the molecular cargo of exosomes represents a rich source for novel cancer biomarker discovery (Sundararajan et al. 2018).

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We provide innovative technologies to speed up your cancer biomarker discovery workflow, including our SMARTer ThruPLEX Plasma-Seq Kit (Figure 1), designed to construct NGS libraries from DNA present within body fluids such as cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). Our chemistry has been optimized to work efficiently with blood samples and is compatible with leading target enrichment platforms for whole-exome sequencing or specific gene panels. Moreover, our SMARTer ThruPLEX technology has been successfully employed and cited by a number of groups, including Murtaza et al., Xia et al., and Patel et al., who demonstrated the applicability of noninvasive monitoring of tumor chemo-resistance by sequencing ctDNA from liquid biopsies in various types of cancers.

SMARTer ThruPLEX Plasma-Seq workflow

Figure 1. SMARTer ThruPLEX Plasma-Seq Kit single-tube workflow. Create indexed libraries starting with 1 to 30 ng of cell-free DNA in three simple steps: end repair, adapter ligation, and high-fidelity library amplification. No purification or sample transfer steps are required. The streamlined workflow is performed in two hours in a single tube or well, preventing sample loss and enhancing positive sample identification.

Tools to accelerate cancer biomarker discovery from exosome molecular cargo, such as microRNAs, include our Capturem Exosome Isolation Kit (Cell Culture), which allows fast (<30 minutes) and easy isolation of exosomes from cell culture media. Our SMARTer smRNA-Seq Kit for Illumina provides a streamlined and fast NGS workflow for the global analysis of small RNAs (smRNAs) from picogram amounts of RNA extracted from exosomes, plasma, and serum. Indeed, Guelfi et al. successfully utilized our SMARTer smRNA-Seq Kit to carry out NGS-based miRNA profiling for noninvasive biomarker discovery in the diagnosis of prostate cancer (Guelfi et al. 2017).


References

Siravegna G. et al. Integrating liquid biopsies into the management of cancer. Nat. Rev. Clin. Oncol. 14, 531–548 (2017).

Sundararajan V. et al. The versatile role of exosomes in cancer progression: diagnostic and therapeutic implications. Cell. Oncol. 41, 223–252 (2018).

Publications citing the use of the SMARTer ThruPLEX-Plasma Seq Kit for noninvasive monitoring of tumor chemoresistance and the SMARTer smRNA-seq Kit for miRNA profiling in prostate cancer diagnosis

Guelfi, G. et al. Next generation sequencing of urine exfoliated cells: an approach of prostate cancer microRNAs research. Sci. Rep. 8, 7111 (2018).

Mayrhofer, M. et al. Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis. Genome Med. 10, 85–98 (2018).

Mouliere, F. et al. Detection of cell‐free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients. EMBO. 12, e9323 (2018).

Murtaza, M. et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112 (2013). 

Patel, K. M. et al. Association of plasma and urinary mutant DNA with clinical outcomes in muscle invasive bladder cancer. Sci. Rep. 7, 5554 (2017).

Xia, Y. et al. Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer. Oncotarget 7, 35818–35831 (2016).

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R400679 ThruPLEX® Plasma-Seq Kit 24 Rxns USD $757.00

License Statement

ID Number  
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 24 reactions.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400679: ThruPLEX Plasma-Seq Kit

R400679: ThruPLEX Plasma-Seq Kit
635723 Capturem™ Exosome Isolation Kit (Cell Culture) 6 Preps USD $366.00

License Statement

ID Number  
273 This product is covered by U.S. Patent Nos. 9,459,188 and/or 10,207,229, exclusively licensed to Takara Bio USA, Inc.

The Capturem Exosome Isolation Kit (Cell Culture) provides a complete solution for the simple and rapid isolation of exosomes from cell culture media. The kit includes six single-use, disposable pre-clearing columns; six single-use, disposable isolation columns; as well as wash and elution buffers. The exosomes are isolated using specially designed Capturem spin columns containing a proprietary, exosome-binding compound which is not antibody-based. Each column will yield up to 2x1011 of purified exosomes in under 30 minutes.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Nanoparticle tracking analysis

Nanoparticle tracking analysis

NTA data of isolated exosomes from HEK293T cells using the Capturem Exosome Isolation Kit (Cell Culture). Panel A. Size and particle concentration distribution plot of exosomes from HEK293T cells (peak at 95 nm). D90 value is shown. Panel B.Three-dimensional graph illustrating size versus intensity (relative frequency of each size range in the eluted sample) versus concentration (particles/ml).

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Comparison of exosomes isolated using the Capturem exosome columns and two other methods

Comparison of exosomes isolated using the Capturem exosome columns and two other methods

Quantitative determination of yield and mean size of exosome isolated through different methods. NTA data showing concentration (Panel A) and size (Panel B) of precleared CM (input) and isolated exosomes (yield) from HEK293T cells using the Capturem kit, precipitation reagent (Competitor T), and ultracentrifugation. The isolation methods are described in the Methods section of the technical note. The exosomes were isolated from 10 ml of precleared CM supernatant for each method. The total exosome yield was divided by input volume to obtain yield (particle/ml of input).

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Western blot analysis for characteristic exosome proteins

Western blot analysis for characteristic exosome proteins

Enrichment of exosomal markers CD81 and Alix in Capturem-isolated exosomes. Western blot analysis of CD81 and Alix as per the protocol described in the Methods section of the technical note. The band densities were calculated using Image Lab software.

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Functional analysis of exosomes

Functional analysis of exosomes

Cellular uptake of exosomes in HEK293T cells. Isolated exosomes from the Capturem kit, ultracentrifugation, and input CM supernatant were labeled with PKH26 dye and uptake studies carried out as described in Methods section of the technical note. The percentage of total yield required for cellular uptake assay is displayed to provide a comparison of the yield differences between the two methods.

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635723: Capturem Exosome Isolation Kit (Cell Culture)

635723: Capturem Exosome Isolation Kit (Cell Culture)
635029 SMARTer® smRNA-Seq Kit for Illumina® 12 Rxns USD $773.00

License Statement

ID Number  
275 SMART-Seq2 Technology. This product is sold under exclusive license from Ludwig Institute of Cancer Research, Ltd. and is covered by US Patent No. 10266894, Japanese Patent No. 6336080, and European Patent No. 3036336, and pending U.S. patent application and/or pending claims of foreign counterparts. For license information, please contact a Takara Bio USA, Inc. licensing representative by phone at 650.919.7320 or by e-mail at licensing@takarabio.com.

The SMARTer smRNA-Seq Kit for Illumina is used to generate small RNA-seq libraries for sequencing on Illumina platforms. This kit was developed to work directly with total RNA or enriched small RNA (including microRNA), for inputs ranging from 1 ng–2 μg. By incorporating features of the SMARTer Stranded RNA-Seq kits, including our proprietary SMART (Switching Mechanism at the 5' end of RNA Template) technology, and primers that include locked nucleic acids (LNAs), this kit allows users to analyze a wide range of smRNA species and generate sequencing libraries of considerable complexity from as little as 1 ng of input material. Illumina adapter and index sequences are incorporated in a ligation-free manner during library amplification, ensuring that diverse smRNA species are represented with minimal bias.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Demonstrating the accuracy of the SMARTer approach for small RNA-seq

Demonstrating the accuracy of the SMARTer approach for small RNA-seq
Demonstrating the accuracy of the SMARTer approach for small RNA-seq. Sequencing libraries were generated from an equimolar pool of 963 synthetic miRNAs (miRXplore Universal Reference) using the SMARTer smRNA-Seq Kit for Illumina (1 ng input; purple), or a small RNA-seq kit from a different vendor (Competitor N) employing an adapter ligation method (100 ng input; blue). Following sequencing, mapping, and counting of reads, miRNA expression levels (Y axis, log scale) were normalized, resulting in an expected expression level equal to 1 for each miRNA, and a 2-fold cutoff was assigned both above and below the expected expression level (indicated by two horizontal lines). For visualization purposes, miRNAs are ranked along the X axis in order of expression level.

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Reproducibility of SMARTer small RNA-seq data

Reproducibility of SMARTer small RNA-seq data
Reproducibility of SMARTer small RNA-seq data. Sequencing libraries were generated in parallel from the indicated input amounts of human brain total RNA using the SMARTer smRNA-Seq Kit for Illumina, and size selected using the BluePippin system. Following sequencing, data processing, and mapping, expression levels of miRNAs identified for each library were quantified and plotted on correlation diagrams. Panel A. Correlation of miRNA expression levels for experimental replicates involving 1 ng inputs. Panel B. Correlation of miRNA expression levels for 2 µg vs. 1 ng inputs.

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Schematic of technology used by the SMARTer smRNA-Seq Kit for Illumina

Schematic of technology used by the SMARTer smRNA-Seq Kit for Illumina
Schematic of technology used by the SMARTer smRNA-Seq Kit for Illumina. SMART technology is used in a ligation-free workflow to generate sequencing libraries for Illumina platforms. Input RNA is first polyadenylated in order to provide a priming sequence for an oligo(dT) primer. cDNA synthesis is primed by the 3’ smRNA dT Primer, which incorporates an adapter sequence (green) at the 5’ end of each first-strand cDNA molecule. When the MMLV-derived PrimeScript Reverse Transcriptase (RT) reaches the 5’ end of each RNA template, it adds non-templated nucleotides which are bound by the SMART smRNA Oligo—enhanced with locked nucleic acid (LNA) technology for greater sensitivity. In the template-switching step, PrimeScript RT uses the SMART smRNA Oligo as a template for the addition of a second adapter sequence (purple) to the 3’end of each first-strand cDNA molecule. In the next step, full-length Illumina adapters (including indexes for sample multiplexing) are added during PCR amplification. The Forward PCR Primer binds to the sequence added by the SMART smRNA Oligo, while the Reverse PCR Primer binds to the sequence added by the 3’ smRNA dT Primer. Resulting library cDNA molecules include adapters required for clustering on an Illumina flow cell (P5 shown in light blue, P7 shown in red), Illumina TruSeq® HT indexes (Index 2 [i5] shown in orange, Index 1 [i7] shown in yellow), and regions bound by the Read Primer 1 or Read Primer 2 sequencing primers (shown in purple and green, respectively). Note that adapters included in the final library add 153 bp to the size of RNA-derived insert sequences.

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635029: SMARTer smRNA-Seq Kit for Illumina

635029: SMARTer smRNA-Seq Kit for Illumina

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