First-strand cDNA synthesis
High-temperature reverse transcription
Total RNA input
Kit size
The ZY Rev Star cDNA Synthesis Kit is a high-efficiency, convenient, contamination-reducing, high-quality first-strand cDNA synthesis kit. It integrates a heat-stable M-MLV GIII reverse transcriptase, RNase inhibitor, dNTPs, Oligo(dT)20VN, and random primers. Simply add the RNA template and nuclease-free water to start the reaction.
The built-in dsDNase can quickly and irreversibly inactivate at reverse transcription temperatures, efficiently removing genomic DNA contamination without EDTA treatment, avoiding qPCR false positives. Whether it is eukaryotic mRNA, or prokaryotic RNA, rRNA/tRNA, and other non-Poly(A) templates, reliable cDNA for qPCR can be stably obtained.
| Product Name | Catalog Number | Specification | Suitable Downstream Applications | Price |
|
ZY Rev Star cDNA Synthesis Kit (Reverse Transcription Kit) |
ZY-RScDNA-100 | 100 reactions （100T） |
Real-Time Fluorescent Quantitative PCR （qPCR / RT-qPCR） |
Inquiry |
The ZY Rev Star Reverse Transcription Kit is based on M-MLV reverse transcriptase and reversely transcribes RNA templates into first-strand cDNA. The 5× premix integrates reverse transcriptase, Oligo(dT)20VN, random primers, dNTPs, and RNase inhibitor, achieving broad template coverage through a dual-primer system; meanwhile, a dsDNase module is introduced to efficiently remove gDNA contamination, supporting both one-step and two-step protocols.
| Steps | Operations |
| 1 Prepare the System | Add 50 ng–1 μg of template RNA, 2 μL of 5X Master Mix, and 0.5 μL of dsDNase on ice, and bring up to 10 μL with Nuclease-Free Water. |
| 2 Mix | Gently pipette up and down to mix and briefly centrifuge. |
| 3 Remove gDNA | Incubate at 37°C for 2 min. |
| 4 First-strand synthesis | Incubate at 55°C for 15 min. |
| 5 Terminate the reaction | Incubate at 85°C for 5 min. |
| 6 Storage | Place on ice for subsequent qPCR; -20°C storage is recommended for no more than 1 week, -80°C can be used for long-term storage, avoid repeated freeze-thaw cycles. |
| Stage | Preparation and Reaction |
| gDNA Removal | Template RNA + dsDNase 0.5 μL + 10× dsDNase Buffer 0.5 μL + water to 5 μL; 37℃ 2 min (can be extended to 5 min if contamination is heavy), 65℃ 2 min Inactivate dsDNase. |
| First-Strand Synthesis | Take 5 μL of the above product + 2 μL of 5X Master Mix (All-in-One) + water to 10 μL; 50℃ for 15 min (if no Poly(A) template, can first incubate at 25℃ for 10 min), 85℃ for 5 min to terminate. |
For templates without Poly(A) structure, it is recommended to use a two-step method to achieve higher yield.
| Components | One-step (10 μL) | Two-step - gDNA removal (5 μL) | Two-step - synthesis (10 μL) |
| Template RNA | 50 ng–1 μg | 50 ng–1 μg | Previous step product 5 μL |
| ZY Rev Star 5X Master Mix | 2 μL | — | 2 μL |
| dsDNase | 0.5 μL | 0.5 μL | — |
| 10× dsDNase Buffer | — | 0.5 μL | — |
| Nuclease-Free Water | Add up to 10 μL | Add up to 5 μL | Add up to 10 μL |
| Reaction conditions | 37℃ 2 min + 55℃ 15 min | 37℃ 2 min → 65℃ 2 min | 50℃ 15 min (without Poly(A), first 25℃ 10 min) |
The 5X Master Mix used for the 'first-strand synthesis' in the two-step method is the component of this kit: ZY Rev Star cDNA Synthesis Master Mix (5X).
RNA extracted from cells often contains residual genomic DNA; if it is not removed before reverse transcription, both gDNA and cDNA will be amplified during downstream qPCR, leading to overestimation of gene expression and distorted results. This kit uses dsDNase to specifically digest double-stranded DNA before reverse transcription.
Traditional DNase I requires additional EDTA to inactivate it, which is cumbersome, and EDTA residues may inhibit reverse transcription; dsDNase in this kit can be rapidly and irreversibly inactivated during the reverse transcription heating step, without the need for EDTA.
For samples with low genomic DNA content, a one-step method is recommended; if the sample is heavily contaminated with gDNA, a two-step method is suggested: first remove gDNA and inactivate dsDNase at 65°C, then perform first-strand synthesis separately.
This kit is primarily designed for qPCR template synthesis, and the product meets the requirements for conventional fluorescence quantification. Because random primers are used, it is not recommended for full-length cDNA cloning of eukaryotes; for cloning purposes, please refer to Zhiqinsheng Biotechnology's cDNA/ORF cloning services.
It is suitable for Poly(A)+ eukaryotic mRNA, as well as prokaryotic RNA, eukaryotic rRNA/tRNA, and other non-Poly(A) templates; small RNAs such as miRNA are not suitable.
cDNA can be used directly for qPCR; for short-term storage, keep at -20°C (recommended no more than 1 week), for long-term storage, keep at -80°C, and avoid repeated freeze-thaw cycles.
This kit uses a thermostable M-MLV GIII reverse transcriptase, supporting high-temperature reverse transcription at 55°C, which can effectively resolve high GC/complex secondary structures and improve reverse transcription efficiency for difficult templates.
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