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| Product Name | One-Step RT-qPCR Master Mix |
| Catalog No. | NATR-HMM-0118 |
| Description | A comprehensive 2× master mix that combines reverse transcription and real-time quantitative PCR into a single-tube workflow. The formulation includes a reverse transcriptase and a hot-start DNA polymerase in a unified buffer system, enabling RNA-to-result detection with SYBR Green I fluorescence readout. |
| Intended Use | Sensitive and specific detection and quantification of RNA targets by real-time RT-qPCR without a separate cDNA synthesis step. Suitable for gene expression profiling, viral RNA detection, and infectious disease testing in research applications. |
| Principle / Technology | Reverse transcriptase converts RNA template into cDNA during an initial incubation step (45–55°C). The hot-start DNA polymerase is then activated at 95°C, and the resulting cDNA is amplified through thermal cycling with SYBR Green I fluorescence detection at each cycle. The single-tube format minimizes handling, contamination risk, and time from sample to result. |
| Detection Method | Real-time fluorescence monitoring with SYBR Green I dye; melt curve analysis for specificity verification; optional probe chemistry with end-user-supplied probes |
| Sample Type | Total RNA, mRNA, in vitro transcribed RNA; viral RNA extracts; cellular RNA from cultured cells and tissues |
| Performance Range / Specifications | RNA input range: 100 fg to 1 μg total RNA per 25 μL reaction; amplicon size: 70–300 bp recommended; dynamic range: 7 orders of magnitude; efficiency: 90–110% for validated primer sets; assay time: approximately 60–90 minutes (40-cycle protocol) |
| Sensitivity / LOD | Reliably detects <10 copies of target RNA per reaction from total RNA background; 1.5-fold discrimination of gene expression changes at Ct <30 |
| Specificity | No detectable amplification in no-template and no-RT control reactions; distinct single melt peak per target; dUTP/UNG carryover prevention optional |
| Reaction Conditions / Protocol | Thaw master mix on ice, set up 25 μL reactions: 12.5 μL master mix, forward and reverse primers (200–400 nM each), template RNA, nuclease-free water to volume; run: 50°C 10 min (reverse transcription), 95°C 2 min (denaturation/RT inactivation), 40 cycles of (95°C 10–15 sec, 60°C 30 sec with fluorescence), melt curve 60–95°C at 0.5°C increments |
| Components / Formulation | Reverse transcriptase, hot-start DNA polymerase, SYBR Green I dye, dNTPs, MgCl2 (optimized), passive reference dye (ROX, instrument-dependent), buffer salts, stabilizers, and enhancers in a 2× master mix formulation |
| Storage Conditions | –20°C protected from light for long-term storage; stable at 2–8°C for up to 3 months; minimize freeze-thaw cycles (<15); set up reactions at room temperature or on ice |
| Shelf Life | 18 months at –20°C from date of manufacture |
| Package Specifications | 200 reactions (1.25 mL × 2, based on 25 μL reaction), 500 reactions (1.25 mL × 5), 1000 reactions (1.25 mL × 10) |
| Product Form | Liquid 2× master mix |
| Quality Control | Each lot tested: one-step amplification of 10 pg to 100 ng HeLa total RNA with GAPDH primers (Ct: 16–26); no amplification in DNase-treated RNA without RT step; linearity confirmed (R² ≥0.99); melt curve single peak; inter-lot consistency verified |
| Key Features | Single-tube RT and qPCR eliminates separate cDNA synthesis; reduces hands-on time by 50% and contamination potential; validated with both SYBR Green and probe detection; suitable for routine gene expression and pathogen screening; compatible with fast cycling conditions on major qPCR platforms |
For research use only, not for clinical use.
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