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| Product Name | AdvanceFast One-Step RT-gDNA Digestion SuperMix for qPCR |
| Catalog No. | RESK-0017 |
| Description | AdvanceFast One-Step RT-gDNA Digestion SuperMix for qPCR is a next-generation one-tube solution that combines genomic DNA elimination and first-strand cDNA synthesis in a single reaction. All components required for reverse transcription are supplied in the 4× One-Step RT SuperMix; simply add the gDNA Remover Mix, template RNA, and water to synthesize first-strand cDNA efficiently while removing genomic DNA contamination. By performing both reactions in the same tube, the product simplifies the workflow, dramatically shortens reaction time, and increases cDNA yield. It is well suited for reverse transcription of complex RNA templates and is particularly effective for low-abundance templates and low-copy-number genes. The resulting cDNA is compatible with both probe-based and dye-based qPCR for high-performance gene expression analysis, but is not intended for long-fragment amplification such as gene cloning. |
| Intended Use | Simultaneous genomic DNA removal and first-strand cDNA synthesis in a single tube, for downstream probe-based or dye-based (SYBR Green) quantitative PCR gene expression analysis. Not intended for long-fragment amplification such as gene cloning. |
| Principle / Technology | A 4× one-step RT SuperMix containing all reverse transcription reagents works together with a gDNA Remover Mix in the same tube: genomic DNA is enzymatically degraded while the reverse transcriptase synthesizes first-strand cDNA from the RNA template. The one-tube design eliminates separate digestion and inactivation steps, shortening the workflow and improving yield. Component A contains a blue tracking dye that visually indicates pipetting progress during reaction setup. |
| Detection Method | Downstream fluorescent quantitative PCR (SYBR Green dye-based or probe-based) |
| Sample Type | RNA templates, including complex RNA templates, low-abundance templates, and low-copy-number gene targets (e.g., human 293T cell RNA, Arabidopsis RNA) |
| Performance Range / Specifications | One-tube completion of gDNA removal and reverse transcription; digestion and reverse transcription step requires only 5 minutes, with the full one-step workflow completed in about 15 minutes. Reliably reverse transcribes 100 pg–1 μg of total RNA. Higher cDNA yields and lower Ct values than comparable one-step RT-gDNA digestion products at both 1 μg and 10 ng total RNA input. cDNA product remains highly stable after 30 freeze-thaw cycles and after 7 days of storage at -20°C, 4°C, 25°C, or 37°C (minimal Ct variation). |
| Sensitivity / LOD | Efficiently reverse transcribes as little as 100 pg of total RNA; verified across genes of 30%–70% GC content over the 100 pg–1 μg input range. |
| Specificity | Built-in gDNA Remover Mix eliminates genomic DNA contamination during the same reaction, ensuring qPCR signals reflect genuine cDNA. If genomic DNA has already been removed during RNA extraction, the gDNA Remover Mix can simply be omitted. |
| Reaction Conditions / Protocol | Add gDNA Remover Mix, template RNA, and water to the 4× One-Step RT SuperMix; gDNA digestion and reverse transcription are completed in a single 5-minute step in the same tube. The reaction volume can be scaled up proportionally according to the manual—remember to adjust the reaction volume setting on the PCR instrument accordingly. |
| Components / Formulation | 4× AdvanceFast One-Step RT SuperMix (blue, with tracking dye) gDNA Remover Mix RNase-free Water |
| Storage Conditions | Store at -25°C to -15°C. |
| Shelf Life | 1 year when stored at -25°C to -15°C. |
| Package Specifications | 10 T 100 T |
| Quality Control | Benchmarked against comparable one-step gDNA-removal reverse transcription reagents from multiple brands using human 293T cell and Arabidopsis RNA: delivers higher cDNA yields and lower Ct values at both 1 μg and 10 ng total RNA input. |
| Expiration Date / Stability | Valid for 1 year at -25°C to -15°C. The cDNA product withstands 30 freeze-thaw cycles and remains stable for 7 days at -20°C, 4°C, 25°C, or 37°C with minimal Ct variation. |
| Compatibility | The cDNA product is compatible with both probe-based and dye-based qPCR; recommended partners include Alta DiagnoTech's universal TaqMan multiplex qPCR master mix and universal qPCR SYBR Green master mix for high-performance gene expression analysis. Because Component A contains a blue tracking dye, the product is not recommended for downstream colorimetric assays such as LAMP, where the dye may interfere with color development. Not suitable for long-fragment amplification such as gene cloning; for that purpose, use Alta DiagnoTech's AdvanceFast 1st Strand cDNA Synthesis Kit. |
| Application Notes / Precautions | Component A is blue and serves as a loading tracker; avoid using the product in downstream colorimetric detection reactions such as LAMP. The gDNA removal step can be omitted if genomic DNA was already eliminated during RNA extraction—simply leave out the gDNA Remover Mix and make up the volume with RNase-free water. The reaction system can be scaled up proportionally as described in the manual; adjust the reaction volume in the thermal cycler program accordingly. |
For research use only, not for clinical use.
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