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| Product Name | Column-Based microRNA Extraction Kit |
| Catalog No. | DREK-0018 |
| Description | A specialized silica membrane spin column kit for the enrichment and purification of microRNA and other small RNA species from cultured cells and tissues. The kit employs a two-step binding strategy: total RNA is first bound to the column, followed by a differential wash step with reduced ethanol concentration that selectively retains small RNA species while allowing larger RNA molecules to pass through. The enriched small RNA fraction includes miRNA, siRNA, piRNA, and other regulatory small non-coding RNAs. |
| Intended Use | Enrichment and purification of microRNA (<200 nucleotides) from cultured cells and animal tissues for miRNA expression profiling by RT-qPCR, microarray analysis, and small RNA sequencing library preparation. |
| Principle / Technology | Samples are lysed in a guanidine isothiocyanate-phenol buffer. After addition of ethanol, total RNA including small RNA species binds to the silica membrane. A specialized wash buffer with optimized ethanol content selectively elutes large RNA species (>200 nucleotides) while retaining small RNA on the column. A final low-ethanol wash removes residual large RNA fragments, and the enriched small RNA fraction is eluted in RNase-free water. |
| Detection Method | UV spectrophotometry; Bioanalyzer Small RNA assay for size distribution and quantitation; NanoDrop for total nucleic acid content; RT-qPCR for specific miRNAs (miR-16, miR-21, miR-122); Agilent Small RNA chip for quality assessment; small RNA-seq library yield and miRNA mapping rate |
| Sample Type | Cultured mammalian cells (fresh pellets), fresh or frozen animal tissues (liver, brain, kidney, heart), tissue stored in RNAlater (after appropriate processing); not recommended for formalin-fixed tissues due to RNA degradation |
| Performance Range / Specifications | Column binding capacity: 30 µg total RNA (small RNA fraction approximately 0.5-5% of total RNA); input: up to 10⁷ cells or 50 mg tissue; small RNA recovery: >70% for miRNA species (miR-16, miR-21 spike-in); enrichment factor: 5-10× concentration of miRNA relative to total RNA; A260/A280: 1.8-2.1 for enriched fraction; size range of enriched fraction: 10-200 nucleotides |
| Sensitivity / LOD | Detects low-abundance miRNAs at <10 copies per cell by RT-qPCR; enriched fraction contains detectable miRNA from as few as 10³ cells; differential expression of miRNAs with fold-change <2 reliably detected |
| Specificity | Enriched fraction contains predominantly small RNA species (10-200 nt); >80% reduction in ribosomal RNA (18S and 28S) compared to total RNA; miRNA recovery validated by detection of miR-16, miR-21, miR-26a, let-7a; co-enrichment of tRNA fragments, piRNA, and other small non-coding RNA species |
| Reaction Conditions / Protocol | Homogenize sample in 700 µL lysis buffer (containing phenol and guanidine isothiocyanate) with β-mercaptoethanol; incubate 5 minutes at room temperature; add 140 µL chloroform; shake vigorously 15 seconds; incubate 3 minutes; centrifuge at 12,000g for 15 minutes at 4°C; transfer aqueous phase; add 1.5 volumes 100% ethanol; mix; transfer to spin column; centrifuge at 8,000g for 30 seconds; apply 700 µL of small RNA wash buffer; centrifuge; apply 500 µL of small RNA wash buffer; centrifuge; wash with 500 µL secondary wash buffer; centrifuge 2 minutes to dry membrane; elute with 30-50 µL RNase-free water; centrifuge; total time: 60-90 minutes |
| Components / Formulation | Lysis buffer (phenol, guanidine isothiocyanate, ammonium thiocyanate, sodium acetate, glycerol), chloroform (user-supplied), 100% ethanol (user-supplied), small RNA wash buffer (Tris-HCl, NaCl, optimized ethanol concentration for selective large RNA removal), secondary wash buffer (Tris-HCl, NaCl, ethanol), RNase-free water, silica membrane spin columns with collection tubes; β-mercaptoethanol (supplied or user-provided) |
| Storage Conditions | Lysis buffer at 2-8°C protected from light; wash buffers at room temperature; columns at room temperature; chloroform stored at room temperature; β-mercaptoethanol at 2-8°C |
| Shelf Life | 18 months for lysis buffer (phenol-based); 24 months for wash buffers and columns; storage at 2-8°C extends lysis buffer stability; check lysis buffer color—discoloration indicates oxidation |
| Package Specifications | 50 preparations and 200 preparations; all reagents included except chloroform and ethanol; β-mercaptoethanol depending on kit format |
| Product Form | Liquid buffers; silica membrane spin columns; lysis buffer containing phenol and guanidine isothiocyanate |
| Quality Control | Each lot validated with HeLa cell small RNA enrichment: miR-21 recovery >70% vs total RNA reference; 18S/28S rRNA reduction >80% by Bioanalyzer; small RNA fraction yields 50-500 ng; A260/A280: 1.8-2.1; RT-qPCR for miR-16 and miR-21 linear across 4-log dilution; DNase/RNase free |
| Key Features | Selective small RNA enrichment without gel purification; enhanced miRNA detection sensitivity; compatible with challenging tissue types; suitable for small RNA-seq library preparation; chloroform extraction step removes proteins and DNA; concentrated eluate for downstream applications |
| Purity | A260/A280: 1.8-2.1 for enriched small RNA fraction; A260/A230: >1.6; residual large rRNA <20% of total as verified by Bioanalyzer |
| Concentration | Elution 30-50 µL; enriched small RNA concentration 1-10 ng/µL; depending on input material; miRNA constitutes approximately 0.01-0.1% of total cellular RNA mass |
| Activity / Unit Definition | Not applicable for RNA enrichment kits |
| Molecular Weight | Not applicable; enriched fraction contains RNAs of 10-200 nucleotides (approximately 3-65 kDa range) |
| Source / Origin | Silica membrane from borosilicate glass microfiber; lysis buffer based on phenol-guanidine isothiocyanate chemistry; all buffer components are molecular biology grade, DEPC-treated or certified RNase-free |
| pH Range / Optimal pH | Lysis buffer pH ~4.0-5.0 (acidic phenol extraction); small RNA wash buffer pH 6.5-7.0; secondary wash buffer pH 7.0-7.5; elution water pH 6.5-7.5 |
| Shipping Conditions | Lysis buffer at 2-8°C with cold packs; wash buffers and columns at ambient temperature; phenol component may require dangerous goods documentation for air transport |
| Expiration Date / Stability | 18 months for lysis buffer at 2-8°C; 24 months for wash buffers and columns; phenol-based lysis buffer should be inspected for color change (pink/red indicates oxidation and reduced effectiveness); accelerated stability testing at 25°C confirms chemistry stability |
| Regulatory / Compliance | For research use only; not for diagnostic procedures; phenol component requires appropriate chemical safety measures and disposal; manufactured in ISO 9001 facility |
| Compatibility | Enriched small RNA fraction suitable for miRNA RT-qPCR (stem-loop or polyA-tailing methods), miRNA microarray analysis, small RNA-seq library preparation (compatible with all major library prep kits), polyacrylamide gel electrophoresis, and Northern blotting for small RNA detection |
| Recommended Buffer System | Acidic phenol-guanidine isothiocyanate extraction; selective ethanol precipitation/differential binding; Tris-buffered wash systems; RNase-free water elution |
| Application Notes / Precautions | Work in a dedicated RNase-free environment; chilled centrifugation at 4°C during phase separation improves RNA integrity; do not disturb the interphase when collecting aqueous layer; the aqueous phase may appear slightly turbid for tissue samples—this is acceptable; ensure columns are completely dry before elution (residual ethanol inhibits downstream enzymatic reactions); avoid repeated freeze-thaw of enriched RNA; for low-input samples, reduce elution volume and use low-retention tubes; for biofluid samples (serum/plasma), this kit requires protocol adjustments |
| Batch-to-Batch Consistency | Between-lot miRNA recovery CV <20% for miR-21 spike-in; size distribution reproducibility verified by Bioanalyzer small RNA chip; column binding capacity controlled >30 µg per column; phenol quality verified per lot by HPLC purity analysis; DNase/RNase testing per batch |
For research use only, not for clinical use.
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