Cat.No.：D14021A
Price: ￥
Specifications：
This product is specifically designed for qPCR experiments. It utilizes the efficient HiFi-MMLV reverse transcriptase to accurately synthesize the first strand cDNA from total RNA or mRNA. The kit is in a pre-mix form, making the operation extremely simple and quick. To address the issue that RNA samples are easily interfered by genomic DNA, this product incorporates a genomic removal module, which can effectively remove residual genomic DNA before reverse transcription, ensuring the accuracy of the quantitative results. Additionally, the cDNA generated by this kit is perfectly compatible with SYBR Green dye method and probe method, meeting the detection requirements of different experimental systems.
Efficient reverse transcription, more reliable qPCR results: The HiFi-MMLV reverse transcriptase has been modified through multiple mutations, significantly reducing RNase H activity and greatly enhancing the extension performance and template affinity; combined with an optimized reaction buffer system for qPCR, it can achieve higher cDNA synthesis efficiency and smaller Ct values.
Built-in genomic clearance module, data reliable and trustworthy: The kit contains an independent genomic clearance component, which can quickly remove residual genomic DNA in the RNA template before reverse transcription, effectively avoiding the interference of genomic DNA on qPCR quantification, and eliminating the need for designing primers across introns.
Simple operation, time-saving and efficient: The pre-mix form is ready to use immediately, just add the template RNA and water to start the reaction; the optimized reaction system can significantly shorten the reverse transcription time and accelerate the experimental process.
High sensitivity, suitable for low-abundance samples: It can efficiently utilize pg-level total RNA templates, providing excellent detection sensitivity for low-abundance transcripts; it also has good reverse transcription effects for RNA templates with complex secondary structures or high GC content.
Flexible primer selection, comprehensive coverage: Two types of primers are provided: Anchored Oligo(dT)₂₃VN and Random Primers. They can be flexibly selected or mixed according to experimental needs, balancing the specific reverse transcription of mRNA and the comprehensive coverage of various RNAs.
Broad product compatibility: The synthesized cDNA can be directly used for SYBR Green dye method and probe-based qPCR, and is also suitable for various downstream applications such as gene expression analysis and miRNA detection.
| Cat. No. | Component | Specification | Quantity |
| B18011A | RNase-free ddH2O | 1 mL | 4 |
| B18021A | Enzyme Mix* | 200 μL | 1 |
| B18031A | 5×All-in-one qRT SuperMix* | 800 μL | 1 |
| B18041A | No RT Control Mix* | 40 μL | 1 |
This product contains thermolabile DNase. Enzyme Mix, 5 × All-in-one qRT SuperMix and No RT Control Mix should be handled on ice. Return to -20℃ storage immediately after use and avoid repeated freeze-thaw cycles.
Store at -30 to -15℃ and transport at ≤0℃.
The cDNA synthesized by this product is suitable for SYBR Green method and probe-based real-time fluorescence quantitative PCR. It can be used for quantitative analysis of gene expression levels in various RNA samples such as cells, tissues, and blood. It is also applicable for miRNA detection, pathogen RNA detection, and gene copy number determination in scenarios involving low-abundance target detection.
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 their advantages, 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 if all components are correctly added and if the enzymes are inactive (replace with new enzymes). Reaction procedure: Confirm if the incubation temperature and time are appropriate; For complex templates, you can increase the temperature or extend the incubation time appropriately.
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.
