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| Product Name | Exosome RNA Isolation Kit, Affinity Membrane, Serum and Plasma |
| Catalog No. | DREK-0033 |
| Description | Affinity membrane-based kit specifically designed for isolation of total RNA (including miRNA, mRNA, lncRNA, and other small non-coding RNAs) from exosomes pre-enriched from serum, plasma, cell culture supernatant, and other biofluids. The kit uses a proprietary hydrophilic affinity membrane that selectively captures RNA from pre-isolated exosome pellets or exosome-enriched fractions. The membrane chemistry minimizes co-purification of proteins, lipids, and DNA, yielding RNA of sufficient purity for RT-qPCR, miRNA profiling, RNA sequencing, and digital PCR applications. The protocol is compatible with exosome preparations obtained via ultracentrifugation, precipitation, size exclusion chromatography, or immunoaffinity methods. |
| Intended Use | Isolation of total RNA from purified exosomes derived from human or animal serum, plasma, urine, saliva, cerebrospinal fluid, and cell culture supernatant for downstream applications including miRNA RT-qPCR, small RNA sequencing, mRNA expression analysis, lncRNA profiling, and exosome biomarker discovery. |
| Principle / Technology | Exosomes are first isolated using the user's preferred method; exosome pellet is lysed in a guanidinium-based buffer that inactivates RNases while releasing RNA; lysate is passed through an affinity membrane spin column that selectively binds RNA; DNA and proteins pass through; bound RNA is washed to remove residual contaminants; pure RNA is eluted in nuclease-free water or low-ionic-strength buffer. |
| Detection Method | Isolate exosomes (ultracentrifugation, precipitation, SEC, or immunoaffinity); resuspend exosome pellet in Lysis/Binding Buffer; vortex vigorously; add ethanol (1.5 volumes); load onto affinity membrane spin column; centrifuge 30 s; wash with Wash Buffer 1; wash with Wash Buffer 2 (2x); elute in 15-30 uL nuclease-free water; centrifuge 1 min. |
| Sample Type | Pre-isolated exosomes from: human serum (0.5-2 mL equivalent), human plasma (0.5-2 mL equivalent), cell culture supernatant (5-20 mL equivalent), urine (10-50 mL equivalent); input exosome amount 1-50 ug total protein. |
| Performance Range / Specifications | RNA yield: 0.1-5 ng/uL (concentration highly variable depending on exosome source, amount, and cargo); RNA size range: enriched for small RNAs (<200 nt including miRNA at ~22 nt) with some mRNA and lncRNA; A260/A280 not reliable for low-concentration exosomal RNA; detectable miRNA by qPCR (Cq typically 25-35 for abundant miRNAs like miR-21, miR-16). |
| Sensitivity / LOD | Capable of recovering exosomal RNA from as few as 10^8 exosome particles (approximately 1 ug exosome protein); miRNA detectable by RT-qPCR from <1 ng total exosomal RNA; suitable for low-input RNA-seq library preparation with 1-10 ng input RNA. |
| Specificity | Affinity membrane binds total RNA (single-stranded, double-stranded, small RNA, mRNA, lncRNA) through hydrophilic interactions; DNA binding is minimal at the ethanol concentration used; protein binding is negligible; the membrane does not distinguish between exosomal and non-exosomal RNA if present in the input sample. |
| Reaction Conditions / Protocol | Exosome lysis: 5-10 min at RT; column binding and washing: 5-10 min total; elution: 1-2 min; total protocol time approximately 20-30 minutes (excluding exosome isolation). |
| Components / Formulation | Lysis/Binding Buffer (guanidinium-based, 25 mL), Wash Buffer 1 (15 mL), Wash Buffer 2 (concentrate, 5 mL — add ethanol before use), Elution Buffer (nuclease-free water, 10 mL), Affinity Membrane Spin Columns (50 columns), Collection Tubes (2 mL), Protocol. |
| Storage Conditions | All components at RT (15-25 C); after adding ethanol to Wash Buffer 2, store at RT with cap tightly closed. |
| Shelf Life | 18 months from date of manufacture. |
| Package Specifications | 20 preparations, 50 preparations. |
| Product Form | Liquid buffers; pre-packed affinity membrane spin columns. |
| Quality Control | Each lot tested for RNA recovery from exosome standard (HCT116 cell culture-derived exosomes): miR-21 detection by RT-qPCR (Cq <=30 from 2 mL culture supernatant equivalent), absence of PCR inhibitors (spike-in control Cq within +/-1 cycle of water control). |
| Key Features | Specifically designed for exosomal RNA (miRNA and small RNA); compatible with all exosome isolation methods; rapid 20-30 min protocol; no phenol extraction; small elution volume (15-30 uL); RNase inhibitor included. |
| Purity | RNA purity: PCR inhibitor-free (spike-in Cq deviation <1 cycle); DNA contamination <0.1% by qPCR; protein contamination below NanoDrop detection threshold. |
| Concentration | Eluted RNA concentration 0.1-5 ng/uL typical (highly source-dependent); elution volume 15-30 uL; total yield 1-150 ng. |
| Activity / Unit Definition | Lysis buffer demonstrates RNase inactivation within 30 seconds of contact (tested with RNase A). |
| Molecular Weight | miRNA: approximately 6,600-8,800 Da (21-23 nt RNA oligonucleotides); mRNA: variable (hundreds to millions of Da). |
| Source / Origin | Affinity membrane: synthetic hydrophilic modified silica matrix; buffers: molecular biology grade reagents; nuclease-free water: DEPC-treated and autoclaved. |
| pH Range / Optimal pH | Lysis/binding buffer pH 5.0-6.0; wash buffer 1 pH 7.0; elution in nuclease-free water pH 6.0-7.0. |
| Shipping Conditions | Ambient temperature. |
| Expiration Date / Stability | 18 months at RT; after opening, use within 6 months; ensure caps tightly closed to prevent ethanol evaporation from Wash Buffer 2. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use; not for clinical biomarker testing. |
| Compatibility | Eluted RNA compatible with: miRNA-specific RT-qPCR (stem-loop or poly-A tailing methods), small RNA library preparation for NGS (NEBNext, TruSeq, QIAseq), Bioanalyzer/TapeStation small RNA analysis, digital PCR (miRNA quantification), and NanoString nCounter miRNA profiling. Do not use RNA for ribosomal RNA depletion workflows as rRNA is not enriched in exosomes. For RNA-seq, use small RNA or total RNA library preparation kits optimized for low-input and degraded RNA. |
| Recommended Buffer System | Lysis/Binding: guanidinium thiocyanate, sodium acetate, ethanol; Wash 1: guanidine HCl, Tris-HCl, ethanol; Wash 2: Tris-HCl, NaCl, ethanol (80%). |
| Application Notes / Precautions | Exosomal RNA yield is inherently low and highly variable between samples, sources, and isolation methods. Always include a spike-in control (e.g., synthetic C. elegans miR-39) to normalize for RNA recovery and RT efficiency. Use the same exosome isolation method consistently within a study to minimize technical variability. Avoid RNase contamination: use filtered pipette tips, wear gloves, and work in a clean area. For precious samples, add an RNase inhibitor (e.g., SUPERase-In) to the elution buffer. Store eluted exosomal RNA at -80 C immediately after isolation; exosomal RNA is susceptible to degradation even at -20 C. |
| Batch-to-Batch Consistency | miRNA recovery within +/-30% of reference lot (due to inherent variability of exosome input); PCR inhibitor test: spike-in Cq within +/-1 cycle; column binding capacity >10 ug total RNA. |
For research use only, not for clinical use.
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