Cat.No.：D14011A
Price: ￥
Specifications：
This product offers a complete reaction system for efficient synthesis of the first strand cDNA from total RNA or mRNA. Its core is equipped with a genetically engineered HiFi-MMLV reverse transcriptase. This enzyme has extremely low RNase H activity, outstanding extension ability, and excellent tolerance to impurities, enabling it to handle RNA templates with complex secondary structures or low abundance with ease. In terms of primer design, the kit uses Anchored Oligo(dT)₂₃VN primers to precisely anchor the Poly(A) tail, effectively ensuring the specificity of the reverse transcription reaction; at the same time, random primers are provided for researchers to choose as needed. The cDNA synthesized by this kit has excellent quality and is fully applicable to the construction of full-length cDNA libraries, routine PCR, and qPCR and other key downstream experiments.
Efficient synthesis, higher yield: HiFi-MMLV reverse transcriptase has been modified through multiple point mutations, with extremely low RNase H activity, significantly enhanced extension performance and template affinity. The cDNA synthesis efficiency and yield are significantly superior to those of similar products.
Ultra-high sensitivity: It can efficiently utilize pg-level total RNA or mRNA for reverse transcription, and has excellent detection sensitivity for low-abundance transcripts.
Strong tolerance to impurities: The optimized enzymatic system has good tolerance to common inhibitors in samples, and is perfectly adapted to crude extracted RNA containing impurities or complex clinical samples.
Unfazed by complex structures: It possesses excellent extension and continuous synthesis capabilities, effectively overcoming the reverse transcription obstacles caused by high GC content and complex secondary structures of RNA.
Flexible primer strategy: It provides Anchored Oligo(dT)₂₃VN and random primers, which can be flexibly combined according to experimental needs, balancing mRNA-specific reverse transcription and full transcriptome coverage.
Minimalist operation and anti-contamination: The pre-mixed design enables immediate use upon opening, significantly simplifying the sample addition process and effectively reducing operational errors and contamination risks.
Extensive downstream applications: The synthesized high-quality cDNA can be directly used in various core experiments such as PCR, qPCR and cDNA library construction.
| Cat. No. | Component | Specification | Quantity |
| B17011A | RNase-free ddH₂O | 1 mL | 1 |
| B17021A | 2 × RT Mix | 1 mL | 1 |
| B17031A | HiScript II Enzyme Mix | 200 μL | 1 |
| B17041A | Oligo(dT)₂₃VN（50 μM） | 100 μL | 1 |
| B17051A | Random hexamers（50 ng/μL） | 100 μL | 1 |
Note: 2×RT Mix contains buffer salts and dNTPs. Mix thoroughly before use. Enzyme Mix contains reverse transcriptase and RNase inhibitor; avoid repeated freeze-thaw cycles. The two primers can be used separately or mixed according to experimental requirements.
Stored and transported at -20℃.
The first chain cDNA synthesized by this product can be widely applied in downstream experiments such as real-time fluorescence quantitative PCR (qRT-PCR), conventional PCR, gene expression analysis, full-length cDNA library construction, and high-throughput sequencing template preparation.
Which types of RNA templates is this kit suitable for?
This kit is applicable to total RNA, Poly(A)+ mRNA, and various RNA samples from different sources (such as RNA extracted from cells, tissues, blood, etc.). The HiFi-MMLV reverse transcriptase, after modification, has a higher tolerance for impurities, and is particularly suitable for low-purity or partially degraded RNA samples, as well as RNA templates with complex secondary structures or high GC content.
How should reverse transcription primers be selected?
The kit provides two types of primers: Anchored Oligo(dT)₂₃VN and Random Primers. You can choose them flexibly according to your experimental needs: Oligo(dT) primers: Specifically bind to the Poly(A) tail of mRNA, suitable for reverse transcription of the majority of eukaryotic mRNAs, especially for full-length cDNA synthesis. Random primers: Can bind to multiple sites of the RNA template, suitable for reverse transcription of RNA without a Poly(A) tail (such as prokaryotic RNA, rRNA, etc.) and the 5' end region of mRNA. Mixed use: Using both types of primers together can combine the advantages of both, improving coverage and cDNA yield.
What is the maximum length of the synthesized cDNA?
The HiFi-MMLV reverse transcriptase exhibits excellent continuous synthesis capability and can successfully synthesize cDNA up to 12 kb in length. For the synthesis of long fragments of cDNA (>5 kb), it is recommended to use Oligo(dT) primers or gene-specific primers, and appropriately extend the reverse transcription reaction time.
Can the reverse transcription products be directly used for qPCR?
OK. The first strand cDNA synthesized by this kit can be directly used as a template for qPCR. It is recommended to appropriately dilute the cDNA product (usually 5-10 times) before adding it to the qPCR system to avoid the inhibition of fluorescence signal caused by high concentration of cDNA.
Does the RNA template need to be pre-denatureed?
For RNA templates with complex secondary structures or high GC content, it is recommended to mix the RNA with the primer and incubate at 65℃ for 5 minutes, then immediately place it on ice for 2 minutes to open the secondary structure of the RNA and improve the primer binding efficiency and the success rate of reverse transcription.
Why did the amplification signal occur in the "No-RT Control" group?
Possible causes include: Genomic DNA contamination: Genomic DNA remaining from the RNA extraction process was amplified by PCR. It is recommended to pre-treat the RNA sample with DNase I or use reverse transcription kits with gDNA removal function. Primers dimerization or non-specific amplification: Check if the qPCR primer design is reasonable. If necessary, redesign the primers. Reagent contamination: Replace with new RNase-free water and repeat the experiment with the primers. Prepare the reaction system in a laminar flow hood to reduce aerosol contamination.
If the yield of cDNA is low or no product is produced, how should one troubleshoot?
The following steps are recommended for troubleshooting: RNA template: Check if the RNA is degraded (using agarose gel electrophoresis); Ensure the RNA concentration and purity are appropriate (with an A260/280 ratio between 1.8 and 2.1); Avoid repeated freezing and thawing of the template. Primers: Check if the primers are degraded; Oligo(dT) primers are not suitable for RNA without a Poly(A) tail. Reaction mixture: Confirm that all components are correctly added and that the enzymes are not inactive (replace with new enzymes). Reaction procedure: Confirm if the incubation temperature and time are appropriate; For complex templates, you can appropriately increase the temperature or extend the incubation time.
Can the reverse transcription products be stored for a long time?
The synthesized cDNA should be stored at -20℃ for a short term (1-2 weeks), and at -80℃ for a long term. Avoid repeated freezing and thawing, and aliquot the samples for storage to reduce the risk of degradation.
