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| Product Name | One-Step RT-gDNA Digestion SuperMix V for qPCR |
| Catalog No. | RESK-0023 |
| Description | The One-Step RT-gDNA Digestion SuperMix V for qPCR performs reverse transcription and genomic DNA removal in a single tube, simplifying handling and reducing the risk of sample contamination and RNA degradation associated with complex pipetting steps. The 5x One Step RT SuperMix contains all reagents required for reverse transcription (fifth-generation reverse transcriptase, RNase inhibitor, Oligo(dT)18 primer, random primer, dNTPs, and buffer); only the gDNA Remover Mix, template RNA, and water need to be added to synthesize first-strand cDNA while eliminating genomic DNA contamination. A 5x One Step No-RT Control SuperMix is also provided to prepare no-reverse-transcriptase controls that verify whether qPCR signal originates from cDNA. The reverse transcriptase tolerates reaction temperatures up to 60°C, suits RNA templates with complex secondary structures, and shows enhanced template affinity for low-input templates and low-copy genes. The cDNA product is compatible with both probe-based and dye-based qPCR; Alta DiagnoTech's universal TaqMan multiplex qPCR master mix and universal SYBR Green qPCR master mix are recommended for high-performance gene expression analysis. |
| Intended Use | One-tube first-strand cDNA synthesis with simultaneous genomic DNA removal for downstream qPCR-based gene expression analysis. |
| Principle / Technology | Single-tube format combining a fifth-generation reverse transcriptase (thermostable up to 60°C, enhanced template affinity) with a gDNA Remover Mix, so reverse transcription and genomic DNA digestion proceed simultaneously. A No-RT control SuperMix enables confirmation that qPCR templates derive from cDNA rather than residual genomic DNA. |
| Detection Method | Reverse transcription followed by probe-based (TaqMan) or dye-based (SYBR Green) qPCR. |
| Sample Type | RNA from human, mouse, Arabidopsis, E. coli, and other samples; suitable for low-input templates and low-copy genes; RNA input from 1 pg to 1 μg. |
| Performance Range / Specifications | Detection of RNA input from 1 pg to 1 μg, outperforming competing products; reverse transcription efficiency (Ct) for genes of different GC contents superior to competing products; digests 200 ng of genomic DNA with lower residual detection Ct than competing products; optimal enzyme inactivation time 30 sec to 5 min; kit stable at 4°C for 14 days; cDNA product stable at 37°C for 14 days and through 50 freeze-thaw cycles. |
| Sensitivity / LOD | Detection of RNA input as low as 1 pg, with detection rates superior to competing products across 1 pg to 1 μg. |
| Specificity | The included No-RT control SuperMix distinguishes cDNA-derived qPCR signal from genomic DNA background; effective gDNA removal (200 ng genomic DNA digested) ensures template specificity. |
| Reaction Conditions / Protocol | Reverse transcription and gDNA digestion are performed in the same tube; the reverse transcriptase tolerates reaction temperatures up to 60°C. Optimal inactivation time is 30 sec to 5 min. |
| Components / Formulation | 5x One Step RT SuperMix: 40 μL (10 T) / 400 μL (100 T) gDNA Remover Mix: 10 μL (10 T) / 100 μL (100 T) 5x One Step No-RT Control SuperMix: 20 μL (10 T) / 40 μL (100 T) RNase-free H2O: 1 mL (10 T) / 2 x 1 mL (100 T) |
| Storage Conditions | Store at -20°C. |
| Shelf Life | 12 months when stored at -20°C. |
| Package Specifications | 10 T, 100 T |
| Product Form | Premix (SuperMix) |
| Quality Control | Kit stable at 4°C for 14 days; reverse transcription product stable at 37°C for 14 days and through 50 repeated freeze-thaw cycles. |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Valid for 12 months at -20°C; cDNA product stable at 37°C for 14 days and through 50 freeze-thaw cycles. |
| Compatibility | Reverse transcription product is compatible with probe-based and dye-based qPCR. Alta DiagnoTech's universal TaqMan multiplex qPCR master mix and universal SYBR Green qPCR master mix are recommended for high-performance gene expression analysis. |
| Application Notes / Precautions | The reverse transcription product is an RNA/cDNA hybrid, not double-stranded DNA, and is therefore less stable than dsDNA. This product is designed for downstream qPCR; the pre-mixed primers favor the short cDNA fragments needed for quantitative PCR, so long-fragment PCR may show non-specific amplification. For PCR or long-fragment PCR, use Alta DiagnoTech's AdvanceFast 1st Strand cDNA Synthesis Kit or its No Dye version. |
For research use only, not for clinical use.
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