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| Product Name | miRNA Universal qPCR SYBR Master Mix (Stem-Loop Method) |
| Catalog No. | RESK-0010 |
| Description | miRNA Universal qPCR SYBR Master Mix (Stem-Loop Method) is a 2× premix for real-time quantitative PCR offering high sensitivity and high specificity, with excellent linearity across a broad quantification range. Its core component, an miRNA-optimized antibody-based hot-start Taq DNA polymerase, is paired with an optimized buffer to effectively suppress nonspecific amplification caused by primer annealing during sample preparation. The premix contains ROX Reference Dye and is compatible with a wide range of qPCR instruments; only primers and template need to be added when assembling reactions. |
| Intended Use | Quantification of microRNAs by stem-loop reverse transcription followed by dye-based qPCR, using cDNA generated with miRNA-specific stem-loop RT primers. Suitable for miRNA expression analysis, including discrimination of closely related family members. For research use. |
| Principle / Technology | Stem-loop method: each miRNA is reverse transcribed with a specific stem-loop RT primer that extends the short miRNA into an amplifiable cDNA, which is then quantified by SYBR Green dye-based qPCR. The antibody-based hot-start Taq DNA polymerase remains inactive during reaction setup, preventing nonspecific amplification from primer annealing, and is activated by heat. ROX Reference Dye is included in the premix (universal ROX) for compatibility with most qPCR instruments. |
| Detection Method | Fluorescent quantitative PCR (SYBR Green dye-based) |
| Sample Type | miRNA cDNA generated by stem-loop reverse transcription (stem-loop RT products) from total RNA; each miRNA requires its own specific stem-loop RT primer. Recommended amplicon length 60-300 bp; annealing temperature 60 °C; suitable for 30%-70% GC content. |
| Performance Range / Specifications | High sensitivity down to 10 pg of total RNA-derived cDNA (validated with 10-fold serial dilutions from 10 pg/µL to 0.1 µg/µL of mouse liver total RNA cDNA amplifying mmu-miR-125a, mmu-miR-132, and mmu-miR-351). Excellent reproducibility: low well-to-well variation across 90 replicate wells (validated with 50-fold-diluted human 293T cell cDNA amplifying hsa-let-7a). Good linearity across a broad quantification range. Outstanding stability: stable for 14 days at 37 °C, through 50 freeze-thaw cycles, and as assembled reactions held at 25 °C for 24 hours. |
| Sensitivity / LOD | Detects as little as 10 pg of starting material (total RNA-derived cDNA), enabling quantification of low-abundance miRNAs. |
| Specificity | Distinguishes single-base differences between members of the same miRNA family (validated with the human hsa-let-7 family, whose members differ by as little as one nucleotide; matching rates calculated by the 2^(-ΔΔCt) × 100% method). |
| Reaction Conditions / Protocol | Reverse transcribe each miRNA separately with its own specific stem-loop RT primer (one miRNA per RT reaction, including the reference gene). Assemble qPCR reactions by adding primers and template to the 2× premix; recommended annealing temperature 60 °C; recommended amplicon length 60-300 bp. Compatible with most qPCR instruments via the built-in universal ROX Reference Dye. |
| Components / Formulation | 2× miRNA Universal qPCR SYBR Master Mix, containing antibody hot-start miRNA Taq DNA polymerase, SYBR Green I, dNTPs, Mg2+, and universal ROX Reference Dye Package sizes: 1 mL; 5× 1 mL; 5× 5 mL |
| Storage Conditions | Store at -20 °C protected from light. Avoid repeated freeze-thaw cycles. The product contains the fluorescent dye SYBR Green I; protect from strong light during storage and reaction setup. |
| Shelf Life | 18 months when stored at -20 °C protected from light. |
| Package Specifications | 1 mL 5× 1 mL 5× 5 mL |
| Product Form | 2× ready-to-use liquid master mix |
| Concentration | 2× |
| Source / Origin | Antibody hot-start miRNA Taq DNA polymerase with optimized buffer, SYBR Green I dye, dNTPs, and universal ROX Reference Dye. |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Stable for 18 months at -20 °C protected from light. The reagent remains stable after 14 days at 37 °C, after 50 freeze-thaw cycles, and assembled reactions remain stable when held at 25 °C for 24 hours. Nevertheless, avoid unnecessary repeated freeze-thaw cycles and protect from strong light. |
| Compatibility | Contains universal ROX Reference Dye; compatible with the vast majority of fluorescence quantitative PCR instruments. |
| Application Notes / Precautions | For studies of multiple miRNAs, design a specific stem-loop RT primer for each miRNA; only one miRNA can be reverse transcribed per RT reaction, and the same applies to the reference gene. Avoid repeated freeze-thaw cycles of the master mix. The product contains the fluorescent dye SYBR Green I; avoid strong light exposure during storage and reaction setup. The mix is validated for stable performance even after 50 freeze-thaw cycles, 14 days at 37 °C, or 24 hours at 25 °C as an assembled reaction, but frozen light-protected storage is recommended for routine use. |
For research use only, not for clinical use.
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