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| Product Name | First-Strand cDNA Synthesis Kit |
| Catalog No. | NATR-HMM-0117 |
| Description | A complete reverse transcription kit for the efficient synthesis of first-strand cDNA from total RNA or mRNA templates. The kit includes a thermostable reverse transcriptase with reduced RNase H activity, a balanced mix of random hexamers and anchored oligo-dT primers, dNTPs, and an RNase inhibitor in an optimized buffer system. |
| Intended Use | Synthesis of first-strand cDNA from total RNA or poly(A)+ mRNA for use as template in PCR, qPCR, cloning, cDNA library construction, and next-generation sequencing library preparation. |
| Principle / Technology | Reverse transcriptase catalyzes the RNA-dependent DNA polymerization reaction, copying RNA into complementary DNA. The kit provides two priming strategies: oligo-dT primers anneal to poly(A) tails of mRNA for selective cDNA synthesis, and random hexamers initiate synthesis throughout the RNA template for complete transcript coverage including 5ʹ ends and non-polyadenylated RNAs. |
| Detection Method | cDNA quantification by UV spectrophotometry or fluorometry; cDNA quality assessment by PCR/qPCR amplification of reference genes; Bioanalyzer or TapeStation analysis of cDNA size distribution; RNA-to-Ct evaluation in RT-qPCR assays |
| Sample Type | Total RNA from tissues, cultured cells, blood, plant material, bacteria, and yeast; mRNA purified by oligo-dT selection; in vitro transcribed RNA; viral RNA |
| Performance Range / Specifications | RNA input range: 1 pg to 5 μg total RNA per 20 μL reaction; cDNA yield: 30–50% conversion efficiency from RNA to cDNA; transcript length coverage: full-length cDNA synthesis for transcripts up to 12 kb |
| Sensitivity / LOD | Enables detection of <1 copy of target RNA per cell by subsequent qPCR from 1 μg total RNA input; differential expression of 1.5-fold reliably detected |
| Specificity | No detectable genomic DNA amplification in RT-negative controls; minimal 3ʹ bias in cDNA representation with random hexamer priming; no detectable RNase activity in reverse transcriptase preparation |
| Reaction Conditions / Protocol | Mix RNA (1 pg–5 μg), primers (oligo-dT or random hexamers), dNTPs, and nuclease-free water; incubate at 65°C for 5 min (RNA denaturation), chill on ice; add RT buffer, RNase inhibitor, and reverse transcriptase; incubate at 25°C 5 min (random hexamer annealing), 42–50°C 30–60 min (cDNA synthesis), 70°C 10–15 min (heat inactivation); store cDNA at –20°C or use directly for PCR |
| Components / Formulation | Reverse transcriptase (200 U/μL, M-MLV-derived, RNase H-minus), 5× reaction buffer (Tris-HCl, KCl, MgCl2, DTT), dNTP mix (10 mM each), oligo-dT primer (50 μM, anchored), random hexamer primer (50 μM), RNase inhibitor (40 U/μL), nuclease-free water |
| Storage Conditions | All components stored at –20°C; enzyme and RNase inhibitor should not be stored at 4°C for more than 24 hours; primers and dNTPs stable for up to 3 months at 2–8°C |
| Shelf Life | 18 months at –20°C from date of manufacture |
| Package Specifications | 100 reactions (20 μL), 200 reactions, and 500 reactions; includes all reagents for cDNA synthesis |
| Product Form | Liquid enzymes, buffers, and primers; frozen shipment recommended |
| Quality Control | Each lot tested: cDNA synthesis from 1 μg HeLa total RNA; qPCR amplification of GAPDH (Ct <20) and PPIA (Ct <22); linearity of cDNA yield across 4-log input range (R² ≥0.98); no-RT control confirms absence of genomic DNA; functional testing across 8 reference genes with PCR efficiency 90–110% |
| Key Features | Dual priming options with oligo-dT and random hexamers in convenient premix; thermostable RT enables synthesis at 50°C for high-GC RNA templates; reduced RNase H activity increases cDNA yield and length; premixed primer/reagent options reduce pipetting steps; compatible with SYBR Green and probe-based qPCR |
For research use only, not for clinical use.
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