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| Product Name | AdvanceFast 1st Strand cDNA Synthesis SuperMix for qPCR (with gDNA Digester) |
| Catalog No. | RESK-0016 |
| Description | AdvanceFast 1st Strand cDNA Synthesis SuperMix for qPCR (with gDNA Digester) is a next-generation, ready-to-use reverse transcription premix optimized for qPCR applications. Compared with the previous generation, it delivers markedly stronger resistance to residual inhibitors, faster reverse transcription, and higher cDNA yields, making it suitable for RNA from animal, plant, and microbial sources. The included gDNA Digester Mix offers substantially enhanced genomic DNA digestion capacity—removing up to 1000 ng of residual DNA—to eliminate genomic contamination in RNA templates and ensure reliable downstream results. The SuperMix contains all components required for reverse transcription; simply add RNA template and RNase-free water to start the reaction, which simultaneously inactivates the gDNA digester and preserves cDNA integrity. The synthesized cDNA is compatible with both dye-based and probe-based qPCR, but is not intended for long-fragment amplification such as gene cloning. |
| Intended Use | Rapid first-strand cDNA synthesis from RNA templates with simultaneous removal of contaminating genomic DNA, for downstream dye-based (SYBR Green) or probe-based quantitative PCR gene expression analysis. Not intended for long-fragment amplification such as gene cloning. |
| Principle / Technology | The premix is built on an engineered reverse transcriptase with enhanced inhibitor tolerance and processivity, enabling efficient cDNA synthesis even from degraded or inhibitor-containing RNA. A dedicated gDNA Digester Mix enzymatically degrades residual genomic DNA (up to 1000 ng) before reverse transcription; the reverse transcription step simultaneously terminates the digester activity to protect cDNA integrity. An optimized blend of random N6 primers and Oligo(dT) primers is pre-included, so no additional primers are required. Reverse transcription is performed at elevated temperature to overcome complex RNA secondary structures. |
| Detection Method | Downstream fluorescent quantitative PCR (SYBR Green dye-based or probe-based) |
| Sample Type | Total RNA from animal, plant, and microbial samples; also effective with degraded RNA and RNA containing residual inhibitors |
| Performance Range / Specifications | Reverse transcription in 5 minutes; entire workflow completed in approximately 7 minutes 5 seconds. Compatible with 10 pg–5 μg RNA input. Linear over 10 pg to 1 μg RNA input with R² up to 0.99; amplification efficiency up to 96.4% (293T cell RNA) and 100% (Arabidopsis RNA). Higher cDNA yields and lower Ct values than comparable products across targets of different GC content and species. Digests up to 1000 ng of residual genomic DNA. Synthesized cDNA is stable for 7 days at room temperature; the reagent withstands up to 30 freeze-thaw cycles. |
| Sensitivity / LOD | Detects as little as 10 pg of RNA. |
| Specificity | The gDNA Digester Mix efficiently removes residual genomic DNA contamination from RNA templates (verified with up to 1000 ng of human 293T cell DNA), ensuring that qPCR signals originate from cDNA rather than genomic DNA and improving result reliability. |
| Reaction Conditions / Protocol | Add RNA template and RNase-free water to the SuperMix (no additional primers required); reverse transcription proceeds at elevated temperature, which minimizes the impact of complex RNA secondary structures. Reverse transcription time: 5 minutes; total protocol time approximately 7 minutes 5 seconds. Follow the recommended program in the manual; extended reverse transcription times are not recommended, as they may delay Ct values. After the 85°C inactivation step, set the thermal cycler to hold at 4°C indefinitely to protect the product from residual heat. |
| Components / Formulation | AdvanceFast SuperMix gDNA Digester Mix -V2 RNase-free H2O |
| Storage Conditions | Store at -25°C to -15°C. The reverse transcription product can be used immediately or stored short-term at 4°C; for long-term storage, aliquot and keep at -20°C, avoiding repeated freeze-thaw cycles. |
| Shelf Life | 1 year when stored at -25°C to -15°C. |
| Package Specifications | 10 T 100 T |
| Product Form | Premix (ready-to-use master mix) |
| Quality Control | Performance verified across RNA templates from multiple species and GC contents, delivering higher cDNA yields and lower Ct values than comparable reverse transcription reagents; linearity (R² up to 0.99) and efficiency (up to 100%) confirmed over the 10 pg–1 μg input range. |
| Expiration Date / Stability | Valid for 1 year at -25°C to -15°C. The reagent tolerates up to 30 freeze-thaw cycles. Synthesized cDNA shows no significant Ct shift (≤0.5 Ct) after 1–7 days at -80°C, -20°C, 4°C, 25°C, or 37°C; opened reagent may be kept at 4°C free from cross-contamination and should be used within 2 weeks. |
| Compatibility | The cDNA product is compatible with both dye-based and probe-based qPCR reagents, including Alta DiagnoTech's qPCR master mixes. Not suitable for downstream long-fragment amplification such as gene cloning; for high-efficiency reverse transcription of long fragments, use Alta DiagnoTech's AdvanceFast 1st Strand cDNA Synthesis Kit. |
| Application Notes / Precautions | No additional primers are needed—the premix already contains optimized random N6 and Oligo(dT) primers for routine RNA qPCR; for small RNA, circular RNA, or long-fragment PCR, use a kit with separate primers such as Alta DiagnoTech's AdvanceFast 1st Strand cDNA Synthesis Kit. Mix the reagent by gentle inversion followed by brief centrifugation; do not vortex vigorously. Compatible RNA input range is 10 pg–5 μg; excessively high RNA input may introduce inhibitory carryover components—dilute the cDNA if Ct values increase or curves become erratic. Do not extend the reverse transcription time beyond the recommended program, as longer times may delay Ct values. After the 85°C inactivation step, program the thermal cycler to hold at 4°C indefinitely so the product is not exposed to heat from the heated lid if left in the instrument. No concentration measurement or adjustment of the RT product is needed before qPCR; residual dNTPs interfere with spectrophotometric readings, and equal input is already controlled by adding equal RNA mass. The cDNA product remains usable after up to one week at room temperature if accidentally left out. Opened reagent stored at 4°C (without pipette cross-contamination) should be consumed within 2 weeks. |
For research use only, not for clinical use.
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