Exosome DNA Extraction Kits Summary Section 5 Introduction 40 EXO-DNAc 41 EXO-DNA 43
Introduction Genomic DNA Extractiom Kits Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ Distributors: www.hansabiomed.eu/index. php/distributors Technical support For informations and techincal support contact us at: info@hansabiomed.eu Circulating DNA is emerging as a novel non-invasive tool for patient s stratification and disease monitoring. While most of the research has focused on circulating cell-free (cfdna) or circulating-tumor-cell-(ctc)-derived DNA, extracellular vesicle- (EVs)-associated DNA (EV- DNA) is emerging as a third valuable liquid biopsy platform. In fact, genomic single or double-stranded DNA and mitochondrial DNA have been recently detected in extracellular vesicles. In particular, the majority of double-stranded DNA seems to be associated with tumor derived exosomes (Thakur BK et al. 2014; Kalhert et al. 2014 ) where it represents the entire genome of the cancerous tumor from which exosomes were derived. This discovery corroborates the potential of exosomes, which can be easily obtained from a simple blood test. HBM-LS is the first company that developed two kits for the isolation of circulating and Exosome-associated genomic DNA: EXO-DNA and the new EXO-DNAc, which includes a DNA concentrator for concentrating the yield (4 fold concentration) and increasing the purity of the DNA to the levels required for digital PCR (dpcr) analysis and Next Generation Sequencing (NGS). EXO-DNAc: Isolation of circulating and Exosome-associated genomic DNA. Optimized protocol for dpcr and NGS. EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA. Suitable for qpcr 40
EXO-DNAc: cfdna and EV-associate DNA isolation kit for dpcr and NGS 5 EXO-DNAc Kit combines the ability of our reagent DNA-Prep to co-isolate EVs and circulating DNA from biofluids (plasma, urine, serum etc..) or culture supernatants with a user friendly system for DNA purification. Isolated vesicles are lysed with the appropriate lysis buffer and DNA is purified by spin columns and optimized buffers with a fast turnaround time (approximatively 30 minutes). Finally, DNA is concentrated with an appropriated spin column to the level required for digital PCR analysis and NGS. * After the isolation step, the pellet containing intact EVs and cfdna can be treated with Dnase to eliminate cfdna. Following Dnase treatment, DNA extraction from microvesicle proceeds with the same protocol (see example at page 46). Cat. Code Description Size EXO-DNAc: Isolation of circulating and EV-associated genomic DNA HBM-DNAc-PS-20/# Isolation of circulating and exosome associated 20 reactions HBM-DNAc-PS-40/# DNA from plasma and serum. HBM-DNAc-UC-20/# Isolation of circulating and exosome associated 20 reactions HBM-DNAc-UC-40/# DNA from urine and cell culture media. Applications Discovering of mutations by digital PCR or qpcr. Direct exosome capture and DNA purification from biofluids of cell media without time consuming purification steps. Isolation and profiling of genomic EV-associated DNA by DNAse treatment. DNA can be used for NGS. Advantages Highly pure circulating and EVs-associated DNA. Fast and easy protocol Small volume amount required. Suitable for concomitant profiling of cell-free circulating DNA and EVassociated DNA. Kit components Description EXO-DNAc: Isolation of circulating and EV-associated genomic DNA DNA-Prep Reagent for exosome isolation. Lysis buffer Buffer for exosome lysis. Washing buffers Two washing buffers for DNA purification in spin column. Elution Buffer Buffer for DNA elution from spin column. Columns and Tubes Spin columns for DNA purification and tubes for DNA collection. DNA concentrator Spin columns for DNA concentration Lyophilized Exosome Standard Exosome standards (1 vial, 100 μg) that can be chosen from the list of available Lyophilized Exosome Standards (Pag 3). 41
EXO-DNAc: performance and applications Concentrator improves the quality of cfdna and EV-DNA, suitable for dpcr and NGS Circulating and EV associated DNA was isolated from different samples, including human plasma, serum and four different cell culture media using both EXO-DNAc and EXO-DNA. DNA yield were finally analyzed by Agilent Bioanalyzer and by Nanodrop showing the same electropherogram profile, but increased concentration (4X) and purity for DNA concentrated with EXO-DNAc protocol. 1. Electropherograms of genomic DNA extracted with EXO-DNA kit following the standards protocol and with EXO-DNAc, which includes the concentration step. 2. EXO-DNAc is able to concentrate 4x the DNA yield extracted with the standard protocol (EXO- DNA). DNA yield measured by Nanodrop. 3. EXO-DNAc improves the DNA purity compared to the standard protocol (EXO-DNA). DNA purity expressed in 260/280 ratio. EXO-DNAc is a flexible platform for detecting actionable mutations in cancer patients by dpcr Detection of the mutation BRAFV600E by digital PCR in genomic DNA of serum of a metastatic prostate cancer patient treated with abiraterone. Circulating and EVs associated DNA was extracted by EXO-DNAc kit and a competitor kit. Digital PCR analysis revealed mutations in DNA extracted with EXO-DNAc, whereas no mutation were detectable in DNA extracted with competitor kit (Validated by Exosomics Siena). 4. Detection of BRAFV600E by dpcr in DNA isolated from serum of a prostate metastatic cancer patient. 5 mutations of BRAF gene were detected in DNA extracted with EXO-DNAc, whereas no mutations were identified with the competitor kit. In addition EXO-DNAc kit shows a decreased background (lower number of BRAF WT). 42
EXO-DNA: Isolation of circulating and Exosome-associated DNA EXO-DNA combine the ability to isolate EVs and ciculating DNA from a wide range of biofluids (plasma, urine, serum etc.) or culture supernatants with a user friendly system of DNA purification. Isolated EVs are lysed with the appropriate lysis buffer and DNA is purified by spin columns and optimized buffers with a fast turnaround time (approximatively 30 minutes). In addition EXO-DNA Kit provides lyophilized exosomes to be used as quality controls for exosome capture and DNA extraction. * Pellet after isolation can be treated with Dnase to eliminate cell free circulating DNA. After Dnase treatment DNA extraction from microvesicle proceeds following the same protocol (see example at page 46). Cat. Code Description Size EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA HBM-DNA-PS-20/# Isolation of circulating and exosome associated 20 reactions HBM-DNA-PS-40/# DNA from plasma and serum. HBM-DNA-UC-20/# Isolation of circulating and exosome associated 20 reactions HBM-DNA-UC-40/# DNA from urine and cell culture media. Kit components Description EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA DNA-Prep Reagent for exosome isolation. Lysis buffer Buffer for exosome lysis. Washing buffers Two washing buffers for DNA purification in spin column. Elution Buffer Buffer for DNA elution from spin column. Columns and Tubes Spin columns for DNA purification and tubes for DNA collection. Lyophilized Exosome Standard Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Pag 3). Applications 5 Purification of circulating and exosome-associated DNA. Direct exosome capture and DNA purification from biofluids of cell media without time consuming purification steps. Isolation of genomic exosome-associated DNA by DNAse treatment. Profiling of exosome associated genomic DNA. Advantages Time saving procedure. The only kit on the market providing Exosome Standards as control. Nucleic acids extracted from a small volume amount. Possibility to profile together circulating and exosomeassociated genes. 43
EXO-DNA: performance and applications Exosome-associate DNA is suitable for point mutation analysis by allele-specific PCR. Healthy donor serum was spiked with 100 μg of purified exosome from BRAFV600E-positive A375 melanoma cell lines. Vesicles were isolated by chemical precipitation with DNA-Prep contained in EXO-DNA kit and treated with or without Dnase 1, to distinguish circulating + Exosome related and only Exosome related DNA. Following digestion, DNA was extracted with EXO-DNA kit and analyzed by bioanalysis and allele-specific qpcr (Fig 5 and 6). 5. Electropherograms of genomic DNA extracted with EXO-DNA Kit with or without Dnase treatment Ct Values (Fluorescence) 24 26 28 30 32 34 36 - DNase + DNase BRAF WT BRAF V600E 6. BRAF WT (wild type) and BRAF V600E amplification by qpcr from DNA extracted with EXO-DNA Kit with or without Dnase treatment. EXO-DNA Kit guarantees high efficiency isolation of circulating and EV-associated DNA Amplification of beta-actin from exosome-derived DNA. Exosomes were isolated from serum with or without artificial spike (A375-derived exosomes) using DNA-Prep solution and treated (or not) with DNAse I. DNA was extracted with HBM EXO- DNA kit and competitor and beta actin was amplified by qpcr. 25 26 Ct Values (Fluorescence) 27 28 29 30 31 32 33 34 35 Dnase A375 A375 DNAse HBM Competitor 3. Beta-actin amplification from exosome-derived DNA, extracted with HBM-LS EXO-DNA Kit and a competitor kit for circulating DNA isolation. 44