Cat.No.：D14031A
Price: ￥
Specifications：
This product is specifically designed for miRNA reverse transcription reactions and adopts the efficient A-tailing principle. In the reaction system, the Poly(A) polymerase first modifies the 3' end of miRNA with a poly(A) tail, and then, in combination with the Universal Oligo(dT) primer, completes reverse transcription, accurately generating the first strand of miRNA cDNA. This kit has integrated the Poly(A) modification and all the necessary reagents for reverse transcription, achieving a truly "one-stop" operation. The simple experimental process not only reduces operational errors but also significantly improves the experimental efficiency of miRNA detection.
High sensitivity: It can efficiently obtain the first strand cDNA corresponding to miRNA from total RNA ranging from 10 pg to 2 μg, and has good detection ability for low-abundance miRNA.
High specificity: The Poly(A) polymerase specifically recognizes single-stranded miRNA, minimizing the reverse transcription of miRNA precursors; in the subsequent qPCR, only specific forward primers need to be designed, combined with a universal reverse primer, to complete the detection.
High throughput and sample saving: One tube of reaction can simultaneously reverse transcribe multiple miRNA, and the cDNA obtained from one reaction can be used for the detection of multiple targets, effectively reducing errors and saving sample volume.
Simple operation: In the form of pre-mix, tailing and reverse transcription can be completed simultaneously in the same tube, simplifying the operation process and reducing human error.
Good product compatibility: The synthesized cDNA can be compatible with SYBR Green staining method and probe-based qPCR, suitable for various downstream applications such as gene expression analysis.
| Cat. No. | Component | Specification | Quantity |
| B19011A | RNase-free ddH2O | 1 mL | 1 |
| B19021A | 2 × miRNA RT Mix | 250 μL | 1 |
| B19031A | HiScript miRNA Enzyme Mix | 50 μL | 1 |
| B19041A | Universal reverse Q primer（10 μM） | 625 μL | 1 |
Note: 2 × miRNA RT Mix contains dNTPs and primers required for reverse transcription. Mix thoroughly before use. HiScript miRNA Enzyme Mix contains Poly(A) polymerase, reverse transcriptase and RNase Inhibitor. It is temperature-sensitive and must be handled on ice; return to -20℃ immediately after use. Universal reverse Q primer is a universal reverse primer, used together with specific forward primers for miRNA qPCR detection.
Store at -20℃ and avoid repeated freezing and thawing.
The cDNA synthesized by this product is suitable for SYBR Green dye method and probe-based qPCR. It can be used for miRNA expression profiling analysis, detection of miRNA expression levels in tissues/cells, and analysis of miRNA differential expression between different samples, etc.
Which types of RNA templates is this kit suitable for?
Suitable for total RNA or enriched miRNA samples. The tailing method can perform non-discriminatory reverse transcription on all miRNAs in the sample, which belongs to high-throughput reverse transcription. For samples with low miRNA content (such as serum, plasma, etc.), after extraction, reverse transcription can be directly carried out without measuring the concentration. The range of template input is 10 pg - 2 μg total RNA.
How does this kit perform miRNA reverse transcription based on what principle?
This kit adopts the principle of the A-tailing method. Firstly, Poly(A) polymerase is used to add a poly(A) tail to the 3' end of the miRNA, enabling it to acquire a Poly(A) tail structure. Then, a universal reverse transcription primer containing Oligo(dT) is combined with the Poly(A) sequence, and under the action of reverse transcriptase, the first strand of the elongated cDNA is synthesized. The tailing and reverse transcription are completed in one step.
What are the characteristics and advantages of tail-adding reverse transcription?
The tailing method enables uniform reverse transcription for all miRNAs in the sample, and one reverse transcription can detect multiple miRNAs. It is suitable for studies involving multiple miRNAs (more than 3), such as the preliminary screening of a large number of miRNAs. With one reverse transcription, multiple target qPCR amplifications can be performed, saving sample volume and being more convenient. For researchers who only need to detect 1-2 miRNAs, they can choose other methods according to their actual needs.
How are the reverse transcription primers for internal reference (such as U6) operated?
The internal control can be reverse transcribed together with the target miRNA. This kit provides forward and reverse primers for U6 internal control, which can complete the reverse transcription together with the target miRNA in the same reaction tube without the need for a separate internal control reaction. During operation, simply add the internal control primers together with the sample RNA according to the recommended system in the manual.
How should the temperature and duration of the reverse transcription reaction be set?
The recommended reaction conditions are incubation at 42°C for 15–30 minutes. After the reaction is completed, it should be incubated at 85°C for 5 minutes to inactivate the enzyme. This kit has combined the addition of Poly(A) tailing and reverse transcription into one step, eliminating the need for separate operations and simplifying the experimental process.
When adding the total RNA, will all the RNA be modified with a tail?
Yes. The tailing method uniformly adds tails to all RNAs (including miRNAs and other small RNAs) in the sample. This is one of the reasons why the tailing method can achieve high-throughput detection. However, it is recommended to use the enriched miRNAs as templates first to further improve the detection specificity.
Can this kit be used for the reverse transcription of regular mRNA?
It is not recommended. This kit is specifically developed for miRNA reverse transcription. The reverse transcription primers are designed differently, and the reverse transcriptase has been genetically modified to have lower efficiency in reverse transcribing longer RNA templates. For the reverse transcription of regular mRNA, please use the dedicated mRNA reverse transcription kit.
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 quality: Check if the RNA is degraded; ensure that the template dosage is within the recommended range (10 pg - 2 μg). Template type: Confirm that the total RNA or enriched miRNA is being used, rather than RNA samples that have already had small RNAs removed. Reaction system: Confirm that all components are correctly added, and that the key enzyme components are operated on ice. Reaction procedure: Confirm that the incubation temperature and time are correct.
What are the differences between this product and the conventional reverse transcription kit?
① This product is specifically designed for miRNA short sequences (21–23 nt). It uses the A-tailing method to add a Poly(A) tail to the miRNA before reverse transcription. ② The tailing and reverse transcription are completed in one step, making the operation simple. ③ One reverse transcription can detect multiple miRNAs, achieving high-throughput detection. ④ It is equipped with a universal reverse primer, and only the design of a specific forward primer is required for qPCR detection.
