Extracellular Vesicle RNA isolation Kits

Transcription

1 Extracellular Vesicle RNA isolation Kits Summary Section 4 Introduction 42 Exo-TotalRNA and TumorExo-TotalRNA isolation kits 43

2 Extracellular Vesicle RNA extraction kits Ordering information Products can be purchased directly in our on-line shop: Distributors: Technical support For informations and techincal support contact us at: Introduction Exosomes shuttle functional RNA molecules in the target cell. Increasing evidence suggests a role for exosome-derived mirnas in the development and/or progression of specific human diseases. Pathogenic mirnas might be exploited as novel therapeutic targets or disease biomarkers in complex diseases, including cancer. In fact, mirnas seem to play critical roles as transcriptional and post-transcriptional regulators of epigenetic mechanisms and cell processes and have been linked to the etiology, progression and prognosis of cancer. Similar mirna expression patterns between tumor tissue samples and circulating exosomes have been observed. HBM-LS has developed optimized solutions for the efficient extraction of highquality total RNA (mirna and mrnas) from the overall exosomes and microvesicle population or from tumor-specific exosome subpopulation, which helps to facilitate the identification of tumor mirna or mrnas signatures from human biofluids or cell culture media. HBM offers three different kits for exosome capture and RNA extraction. EXO-TotalRNA: Overall Exosome Immunocapture and RNA extraction kit: Kit allows, by immunocapture, the isolation of overall exosome population from human biofluids or cell culture media and RNA extraction. TumorEXO-TotalRNA: Tumor Exosome Immunocapture and RNA extraction kit: Kit allows, by immunocapture, the enrichment of tumor-specific exosome population from human biofluids and RNA extraction. RNA Basic Kit: Kit allows RNA extraction from exosomes pre-isolated with different methods (ultracentrifugation, chemical precipitation, immunocapture, size-chromatography etc.) 42

3 Catalog 2018 Section 4 Extracellular Vesicles RNA extraction kits Exo-TotalRNA and TumorExo-TotalRNA isolation kits Exo-TotalRNA and TumorExo-TotalRNA are the only kits for exosome RNA isolation on the market which use immunobeads for capturing vesicles. HBM-LS immunobeads can capture the overall exosome population from a wide range of media, including cell culture supernatants and human biofluids (plasma, serum, urine, etc.) as well as enrich some specific exosome subpopulations (tumorderived exosomes). The captured exosomes are subsequently lysed with an optimized lysis buffer and total RNA is purified using spin columns with a fast and user-friendly process. Eluted RNA can be used for downstream analyses or stored at -80 C. Exosome standards for positive control are also included in the kit. All our kits guarantee high specificity for exosomal RNA and high yield of total RNA (including small RNAs). Applications Direct capture and exosome RNA extraction from human biofluids and cell culture media without initial exosome purification step. Simultaneous mirna and mrna profiling (qrt-pcr, RT-PCR, microarray). Enrichment of tumor-derived exosomes for cancer mirna marker profiling. Advantages High yield of total RNA (including small RNAs). Fast and user-friendly protocol. Small starting amount of sample (less than 1 ml). Better RNA yield than similar products. Cat. Code Description Size EXO-TotalRNA Overall Exosome Immunocapture and RNA extraction Kit (from human biofluids and cell media) HBM-RNA-BOF-20/# RNA extraction from overall exosome population captured with immunobeads 20 reactions TumorEXO-Total RNA Tumor-derived Exosome Immunocapture and RNA extraction Kit (from human biofluids) HBM-RNA-BTF-20/# RNA extraction from tumor-derived exosome population captured with immunobeads 20 reactions The only kit on the market providing Exosome Standards as control. RNA Basic Kit* HBM-RNA-B25 RNA extraction from pre-isolated exosome 25 reactions HBM-RNA-B50 50 reactions *RNA basic kit does not contain immunobeads for exosome isolation. It is designed for isolating the total RNA (mirna + mrnas) from pre-isolated exosomes or extracellular vesicles. Compatible with exosomes isolated via ultracentrifuge, chemical precipitation, size chromatography, immunocapture etc. 43

4 Extracellular Vesicle RNA extraction kits Amount of starting material Whole human plasma and serum can be directly used for RNA extraction. Minimum volume required is 100 μl for plasma and 500 μl for serum. Using urine and cell media, it is recommended to concentrate the sample 10 folds, before incubating the samples with immunobeads. Shipment and storage Kit is shipped at 4 C and all reagents must be stored at 4 C. Kit components Description EXO-TotalRNA Overall Exosome Immunocapture and RNA extraction Kit (from human biofluids and cell media) Immunobeads Immunobeads for overall exosome capture from human biofluids or cell culture media. Beads Washing buffer (5X) Buffer for washing Immunobeads after exosome capture. Lysis buffer Buffer for exosome lysis. RNA Washing buffer Buffer for RNA washing in spin column. Elution Buffer Buffer for RNA elution from spin column. Columns and Tubes Spin columns for RNA purification and tubes for RNA collection. Lyophilized Exosome Standard Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Page 3). TumorEXO-Total RNA Tumor-derived Exosome Immunocapture and RNA extraction Kit Immunobeads Immunobeads for tumor-derived exosome capture and enrichment from human biofluids. Beads Washing buffer (5X) Buffer for washing Immunobeads after exosome capture. Lysis buffer RNA Washing buffer Elution Buffer Columns and Tubes Lyophilized Exosome Standard Lysis buffer RNA Washing buffer Elution Buffer Columns and Tubes Buffer for exosome lysis. Buffer for RNA washing in spin column. Buffer for RNA elution from spin column. Spin columns for RNA purification and tubes for RNA collection. Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Pag 3). RNA Basic Kit Buffer for exosome lysis. Buffer for RNA washing in spin column. Buffer for RNA elution from spin column. Spin columns for RNA purification and tubes for RNA collection. 44

5 Catalog 2018 Section 4 Extracellular Vesicles RNA extraction kits Immunoaffinity EV capture and high quality of EV-derived RNAs from small volumes of sample HD#1 HD#2 Competitor Kit EXO-Total RNA Kit (HBM-LS) 1. Electropherograms of small RNA extracted with HBM EXO-Total RNA kit and Competitor (Agilent 2100 Bioanalyzer) Efficiency of EXO-TotalRNA kit was tested vs a competitor kit for RNA extraction from plasma-derived exosomes. Extraction of total exosome RNA was performed from 100 μl of healthy donor plasma (HD #1 an #2) with Competitor Kit or with the EXO- TotalRNA Kit (HBM-LS). RNA quality was evaluated by electopherogram (Fig 1) with Small RNA microfluidic chips (Agilent 2100 Bioanalyzer). RNA yield was quantified by Nanodrop (Fig 2) and extracted RNA was subsequently retrotranscribed using the miscript II RT kit (Qiagen). mir-21 and b-actin markers were amplified by qpcr (Fig 3 and 4). 600 Total RNA yield (ng) 24 PCR amplification mirna RNA ng Ct Values HBM Competitor 28 HBM Competitor 2. Nanodrop quantification of total RNA yield 3. mirna 21 amplification by qpcr 4. β-actin amplification by PCR 45

6 Extracellular Vesicle RNA extraction kits TumorEXO-Total RNA allows enrichment of cancer-derived mirna from plasma samples Total exosome-derived RNA was extracted from 100 μl of plasma from a healthy donor and a cancer patient using RNA kit EXO-Total RNA (HBM- RNA-BOF) for immunocapture of overall exosomes and RNA extraction or TumorEXO-Total RNA (HBM-RNA-BTF), for tumor-derived exosome enrichment and RNA extraction, respectively. Total RNA extracted was retrotranscribed using the miscript II RT kit (Qiagen) and the expression level of mir-21 and mir-16 were measured relative to control mir451 by qpcr. Usage of TumorEXO-Total RNA shows enrichment in mir-21 and mir-16 in cancer patient (Fig 5 and 6). Fold change (normalized to mir-451) Fold Change (normalized to mir-451) mir-21 HBM-RNA-BOF 7 HBM-RNA-BTF No Cancer Cancer 5. Enrichment in mir-21 expression level in cancer when TumorEXO-Total RNA kit is used. mir-16 HBM-RNA.BOF 7 HBM-RNA-BTF No Cancer Cancer 6. Enrichment in mir-16 expression level in cancer when TumorEXO-Total RNA kit is used. 46