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| Product Name | High-Capacity cDNA Reverse Transcription Kit |
| Catalog No. | NATR-HMM-0138 |
| Description | A complete reverse transcription kit featuring a high-capacity reverse transcriptase enzyme and an optimized buffer containing both random hexamers and oligo-dT primers at balanced concentrations. This dual-priming approach maximizes cDNA yield and provides comprehensive transcriptome coverage from a broad range of RNA input amounts. |
| Intended Use | Synthesis of first-strand cDNA from total RNA for quantitative gene expression analysis by real-time qPCR, TaqMan gene expression assays, SYBR Green qPCR, microarray target preparation, and medium-throughput cDNA archive generation for multi-gene expression studies. |
| Principle / Technology | The high-capacity reverse transcriptase (a recombinant M-MLV derivative with reduced RNase H activity) catalyzes RNA-dependent DNA polymerization using a balanced mix of random hexamers and oligo-dT primers. Random hexamers initiate synthesis throughout transcripts ensuring 5ʹ-end coverage, while oligo-dT primers provide mRNA selectivity. The enzyme's reduced RNase H activity preserves RNA templates during long reactions, increasing full-length cDNA yields up to 7 kb. |
| Detection Method | Real-time qPCR with fluorogenic probe or intercalating dye detection; comparative Ct (ΔΔCt) method with endogenous control normalization; cDNA yield quantification by PicoGreen fluorescence or UV absorbance; quality assessment by successful amplification of reference genes spanning multiple abundance levels |
| Sample Type | Total RNA from mammalian cells and tissues, whole blood, cultured human and animal cell lines, microdissected tissue samples, and purified mRNA; RNA inputs from 10 ng to 5 μg per 20 μL reaction; compatible with RNA extracted by TRI reagent, silica column, and phenol-chloroform methods |
| Performance Range / Specifications | RNA input range: 10 ng to 5 μg total RNA per 20–100 μL reaction (linear across full range); maximum cDNA product length: >7 kb; recommended cDNA synthesis time: 30–120 minutes at 37°C; cDNA synthesis efficiency: >90% conversion at 1 μg input; 5ʹ/3ʹ coverage ratio: <3 for GAPDH and ACTB transcripts |
| Sensitivity / LOD | Detects single-copy transcripts from as little as 10 ng total RNA input (Ct <35); 2-fold gene expression difference reliably detected (Student's t-test P<0.05, n=3) across 4-log RNA input range |
| Specificity | cDNA synthesis from mRNA (polyadenylated) template; minimal gDNA background when RNA samples are DNase-treated prior to RT; no detectable non-specific reverse transcription products in no-RT control reactions by 40-cycle PCR |
| Reaction Conditions / Protocol | Prepare 2× RT master mix on ice (buffer, dNTPs, primer mix, enzyme, water), add RNA sample (10 ng – 5 μg), mix gently, centrifuge briefly; thermal cycler program: 25°C 10 min (random priming), 37°C 120 min (reverse transcription), 85°C 5 min (enzyme inactivation), hold at 4°C; dilute cDNA 1:5 to 1:20 with nuclease-free water before qPCR; for RNA input <100 ng, use undiluted or 1:2 dilution |
| Components / Formulation | High-capacity reverse transcriptase (50 U/μL), 10× RT buffer (Tris-HCl, KCl, MgCl2), dNTP mix (25×, 100 mM total), 10× RT random primer/oligo-dT mix, RNase inhibitor (20 U/μL), nuclease-free water |
| Storage Conditions | All components stored at –20°C; enzyme and RNase inhibitor must remain at –20°C until use; freeze-thaw cycles should be limited to 10; primers and dNTPs may be stored at 2–8°C for up to 3 months |
| Shelf Life | 24 months at –20°C from date of manufacture |
| Package Specifications | 200 reactions × 20 μL, 1000 reactions × 20 μL, and 5000 reactions × 20 μL |
| Product Form | Liquid reagents: enzyme, buffer, dNTP mix, primer mix, RNase inhibitor, water |
| Quality Control | Each lot tested: cDNA yield from 1 μg HeLa RNA generates Ct ≤20 for GAPDH (by qPCR); 5ʹ/3ʹ coverage ratio ≤3 for GAPDH and ACTB; linear response: Ct difference of 3.3 ± 0.3 across 10-fold dilution (R² ≥0.98); no genomic DNA amplification in no-RT control; functional across human, mouse, rat, zebrafish, and Drosophila RNA samples |
| Key Features | Balanced random hexamer and oligo-dT priming provides maximum coverage; broad RNA input range (10 ng to 5 μg) in single protocol; reduced RNase H activity for longer cDNA products; compatible with all commercial qPCR master mixes; pre-optimized single-tube master mix reduces variability |
For research use only, not for clinical use.
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