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| Product Name | FFPE Tissue RNA Extraction Buffer, Xylene-Free Protocol |
| Catalog No. | ATR-SPS-0030 |
| Description | Xylene-free deparaffinization and RNA extraction buffer system for efficient recovery of RNA from formalin-fixed paraffin-embedded (FFPE) tissue sections. FFPE tissues present unique challenges for RNA extraction due to formalin-induced cross-linking and RNA fragmentation. This buffer system employs a paraffin-melting mineral oil emulsion (non-toxic, xylene-free) for deparaffinization, followed by a proteinase K digestion buffer optimized for cross-link reversal at elevated temperature (56-60 °C). The resulting RNA is suitable for RT-qPCR, microarray, and targeted RNA sequencing applications with typical fragment sizes of 100-400 nucleotides depending on fixation conditions and tissue age. |
| Intended Use | Efficient extraction of RNA from archival FFPE tissue sections (5-20 µm thickness) for retrospective gene expression studies, biomarker validation, molecular pathology, and clinical cohort analysis using RT-qPCR or targeted NGS. |
| Principle / Technology | Non-toxic mineral oil emulsion deparaffinization avoids hazardous xylene; proteinase K digestion at 56 °C reverses formalin cross-links and releases RNA; optimized digestion time balances RNA recovery vs. degradation; RNA purified by silica column or magnetic bead downstream protocol. |
| Detection Method | Scrape tissue section into tube; add deparaffinization buffer; incubate at 70 °C for 3 min; centrifuge to separate aqueous and organic layers; collect tissue pellet; add proteinase K digestion buffer; incubate at 56 °C for 15 min (or 60 °C for highly cross-linked older samples); proceed to silica column RNA purification. |
| Sample Type | FFPE tissue sections (5-20 µm thickness, 1-10 sections depending on tissue area); FFPE tissue cores (1-2 mm diameter). |
| Performance Range / Specifications | RNA yield: 0.5-5 µg RNA per 10 µm × 1 cm2 FFPE section depending on tissue type and fixation; typical fragment size 100-400 nt; RNA suitable for amplicons up to 200 bp by RT-qPCR. |
| Sensitivity / LOD | RNA recovered from as few as 1-2 FFPE sections of 10 µm thickness; detectable gene expression by RT-qPCR (Ct <35) for moderately expressed genes from 50 ng FFPE RNA. |
| Specificity | Proteinase K digestion specifically reverses formaldehyde-induced protein-nucleic acid cross-links; deparaffinization removes paraffin embedding matrix; both steps preserve RNA integrity to maximum extent possible for FFPE samples. |
| Reaction Conditions / Protocol | Deparaffinization: 70 °C, 3 min; proteinase K digestion: 56 °C, 15 min (standard) or 60 °C, 30 min (highly cross-linked); can be extended to overnight at 56 °C for larger tissue pieces. |
| Components / Formulation | Deparaffinization Buffer (mineral oil emulsion, xylene-free, 10 mL), Proteinase K Digestion Buffer (5 mL), Proteinase K solution (20 mg/mL, 0.5 mL), DNase I (RNase-free, for optional DNA removal, 0.1 mL), detailed protocol. |
| Storage Conditions | Store Deparaffinization Buffer at room temperature; Proteinase K solution at -20 °C; Digestion Buffer at 2-8 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (sufficient for ~50 FFPE samples), 5 kits. |
| Product Form | Deparaffinization Buffer: two-phase liquid (shake before use); Proteinase K: clear liquid. |
| Quality Control | Each lot tested for RNA yield from standardized human FFPE tissue sections (tonsil or breast carcinoma), RT-qPCR performance for ACTB (amplicon 100-150 bp), and DNA contamination levels. |
| Key Features | Xylene-free non-toxic deparaffinization; optimized proteinase K digestion for FFPE; mineral oil emulsion replaces hazardous organic solvents; compatible with downstream silica column or magnetic bead purification; suitable for archival tissue samples; validated for RT-qPCR and targeted RNA-seq. |
| Purity | Proteinase K recombinant, ≥30 U/mg, DNase/RNase-free; mineral oil USP grade. |
| Concentration | Deparaffinization Buffer: ready-to-use; Proteinase K: 20 mg/mL; use 20 µL per digestion. |
| Activity / Unit Definition | Proteinase K activity: ≥600 U/mL at 37 °C; 1 unit hydrolyzes 1 µmol tyrosine from casein per minute. |
| Molecular Weight | Proteinase K: ~28.9 kDa. |
| Source / Origin | Recombinant Proteinase K from Tritirachium album expressed in Pichia pastoris; pharmaceutical grade mineral oil. |
| pH Range / Optimal pH | Digestion Buffer pH 7.5-8.0. |
| Shipping Conditions | Ambient temperature (Proteinase K on cold pack recommended). |
| Expiration Date / Stability | 12 months at recommended storage; Proteinase K stable for 6 months at 2-8 °C after opening. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. |
| Compatibility | Compatible with FFPE tissue sections from any species. Deparaffinization valid for paraffin with melting point 52-60 °C. Proteinase K digestion compatible with most commercial silica column RNA purification kits (add binding buffer after digestion). For magnetic bead-based purification, ensure bead binding buffer contains sufficient chaotrope. |
| Recommended Buffer System | Proteinase K Digestion Buffer: Tris-HCl with SDS, EDTA, and NaCl, pH 8.0. |
| Application Notes / Precautions | For older or over-fixed FFPE blocks (>5 years), extend proteinase K digestion to 30-60 min and consider adding a second proteinase K spike at 30 min. Design RT-qPCR primers for amplicons of 70-150 bp to accommodate FFPE RNA fragmentation. Include a no-reverse transcriptase control to detect residual genomic DNA. Avoid sections exposed to air for extended periods as RNase contamination increases. The mineral oil layer after deparaffinization can be removed with a clean pipette tip — careful not to disturb the tissue pellet. |
| Batch-to-Batch Consistency | Deparaffinization efficiency >95% wax removal; proteinase K specific activity within ±20% of reference; RNA yield from standard FFPE section within ±30% of reference lot. |
For research use only, not for clinical use.
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