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| Product Name | Column-Based Total RNA Extraction Kit |
| Catalog No. | DREK-0013 |
| Description | A silica membrane spin column kit for the purification of high-quality total RNA from cultured cells and animal tissues. The guanidine isothiocyanate-based lysis buffer with β-mercaptoethanol provides immediate and complete inhibition of endogenous RNases, while the on-column DNase I digestion step eliminates genomic DNA contamination. The kit delivers RNA with high integrity suitable for sensitive gene expression analysis applications. |
| Intended Use | Extraction of total RNA from cultured mammalian cells, animal tissues, and blood leukocyte fractions for RT-PCR, RT-qPCR, cDNA library construction, Northern blotting, and RNA sequencing library preparation. |
| Principle / Technology | Samples are lysed and homogenized in a guanidine isothiocyanate-phenol buffer containing β-mercaptoethanol, which denatures proteins and inactivates RNases. Ethanol is added to adjust binding conditions, and RNA selectively binds to the silica membrane. An on-column DNase I treatment removes genomic DNA. After wash steps with ethanol-containing buffers, pure total RNA is eluted in RNase-free water. |
| Detection Method | UV spectrophotometry (A260/A280 and A260/A230); denaturing agarose gel electrophoresis; Bioanalyzer RNA Nano assay for RIN determination; NanoDrop or Qubit for concentration; RT-qPCR of reference genes for functional quality |
| Sample Type | Cultured mammalian cells (HeLa, HEK293, CHO, Jurkat, primary cells, etc.), fresh animal tissues (liver, kidney, spleen, brain, heart, lung, muscle), white blood cell pellets from whole blood fractionation, yeast cells (with appropriate pretreatment) |
| Performance Range / Specifications | Column binding capacity: 100 µg total RNA; input: up to 5 × 10^6 cells or 30 mg tissue; typical yield from 10^6 HeLa cells: 10-30 µg; from 10 mg mouse liver: 20-60 µg; A260/A280: 1.9-2.1; A260/A230: >1.8; RIN: >7.0 (depending on sample handling) |
| Sensitivity / LOD | Recovers total RNA from as few as 100 cells with micro-scale protocol; detects mRNA transcripts present at <5 copies per cell by RT-qPCR; efficient recovery of RNA from partially degraded samples |
| Specificity | Recovers all RNA species >200 nucleotides; small RNAs (including miRNA) recovered with >50% efficiency; no residual genomic DNA detectable by 35-cycle PCR with intron-spanning GAPDH primers; no detectable RNase activity in eluted RNA |
| Reaction Conditions / Protocol | Disrupt sample in 350-600 µL lysis buffer with β-mercaptoethanol (tissue: homogenize first); add equal volume 70% ethanol; mix; transfer to spin column; centrifuge at 8,000g for 30 seconds; apply 350 µL wash buffer RW1; centrifuge 30 seconds; apply DNase I mixture (80 µL per column); incubate 15 minutes at room temperature; apply 350 µL wash buffer RW1; centrifuge; apply 500 µL wash buffer RPE; centrifuge; repeat RPE wash; dry spin 2 minutes at full speed; elute with 30-50 µL RNase-free water; centrifuge 1 minute; total time: 45-60 minutes |
| Components / Formulation | Lysis buffer (guanidine isothiocyanate, sodium citrate, N-lauroylsarcosine, β-mercaptoethanol), wash buffer RW1 (guanidine hydrochloride, Tris-HCl, ethanol), wash buffer RPE concentrate (Tris-HCl, NaCl, ethanol), DNase I (lyophilized, RNase-free), DNase incubation buffer (Tris-HCl, MgCl2), RNase-free water, silica membrane spin columns with collection tubes |
| Storage Conditions | Lysis buffer at room temperature protected from light; DNase I lyophilized at -20°C, reconstituted at 2-8°C for up to 2 weeks; wash buffers at room temperature; spin columns at room temperature; all components RNase-free certified |
| Shelf Life | 24 months for columns and buffers; DNase I lyophilized: 24 months at -20°C; reconstituted DNase I: 2 weeks at 2-8°C; β-mercaptoethanol: use immediately or replace monthly if opened |
| Package Specifications | 50 preparations and 250 preparations; includes DNase I, all buffers, and spin columns; β-mercaptoethanol supplied or user-provided depending on kit format |
| Product Form | Silica membrane spin columns with collection tubes; liquid buffers; lyophilized DNase I with reconstitution buffer; β-mercaptoethanol vial |
| Quality Control | Each lot tested with HeLa cell RNA: yield ≥15 µg/10^6 cells; A260/A280: 1.9-2.1; RIN ≥8.0; gDNA removal confirmed; RT-qPCR efficiency 90-110% for GAPDH and ACTB; DNase/RNase free; endotoxin <0.05 EU/µL eluate |
| Key Features | On-column DNase digestion for convenient DNA removal; RNase-inhibiting lysis buffer chemistry; 45-minute complete protocol; broad elution volume range; validated for downstream NGS and microarray applications; ready-to-use reagents |
| Purity | A260/A280: 1.9-2.1; A260/A230: >1.8; gDNA contamination below qPCR detection (Ct >35); no protein detectable by Bradford |
| Concentration | Elution volume 30-50 µL; RNA concentration 200-600 ng/µL for cultured cells; 400-1000 ng/µL for tissue samples; total yield sample-dependent |
| Activity / Unit Definition | Not applicable for RNA extraction kits |
| Molecular Weight | Not applicable; total RNA comprises species from ~20 nucleotides to >10 kb |
| Source / Origin | Silica membrane from borosilicate glass microfiber; DNase I from bovine pancreas, chromatography purified to remove RNase; all buffer components molecular biology grade, DEPC-treated where appropriate |
| pH Range / Optimal pH | Lysis buffer pH 6.0-6.5; wash buffer RW1 pH 6.5-7.0; wash buffer RPE pH 7.0-7.5; DNase incubation buffer pH 7.5-7.8; elution water pH 6.5-7.5 |
| Shipping Conditions | Ambient temperature for buffers and columns; DNase I shipped on dry ice; β-mercaptoethanol requires dangerous goods declaration for air transport |
| Expiration Date / Stability | 24 months for columns and buffers; DNase I lyophilized stable 24 months at -20°C; accelerated stability testing at 37°C for 14 days confirms ≥18-month shelf life at recommended storage |
| Regulatory / Compliance | For research use only; not intended for diagnostic use; RNase-free certification per lot; manufactured in compliance with ISO 9001 |
| Compatibility | Purified RNA compatible with all major RT-PCR, RT-qPCR, microarray, and RNA-seq platforms; eluted RNA ready for poly-A selection, ribosomal RNA depletion, and small RNA enrichment; Vacuum manifold compatible |
| Recommended Buffer System | Guanidine isothiocyanate-based lysis and binding; guanidine hydrochloride-containing wash; ethanol-based secondary wash; RNase-free water elution |
| Application Notes / Precautions | Work in a dedicated RNA workspace with RNase-free consumables; add β-mercaptoethanol to lysis buffer immediately before use in fume hood; for tissue, complete homogenization is critical for yield; do not exceed column binding capacity; for cells, wash PBS to remove serum before lysis; avoid over-drying column membrane; if not used immediately, store RNA at -80°C |
| Batch-to-Batch Consistency | Between-lot RNA yield CV <12%; RIN variation <0.5 between lots for HeLa cell control; DNase I activity controlled to ensure complete gDNA digestion per lot; buffer pH within ±0.1 specification; column binding capacity >100 µg per column verified per lot |
For research use only, not for clinical use.
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