Cat.No.：D15031A
Price: ￥
Specifications：
This product is a miRNA-specific qPCR premix developed based on the SYBR Green I hybrid fluorescence method. The kit integrates all the core components required for quantitative reactions in a highly integrated manner. Its core is equipped with antibody-modified hot-start DNA polymerase, and is compounded with a buffer system deeply optimized for miRNA detection characteristics. This system can effectively ensure high specificity and high sensitivity of the quantitative reaction. In addition, the premix is specially designed with a blue trace function, which greatly enhances the convenience and accuracy of micro-sample addition operations.
High-sensitivity quantification: It can stably detect the target miRNA in total RNA samples at the pg level, and maintains a good linear relationship within a wide quantitative range, ensuring accurate and reliable data.
Strong specificity recognition: Relying on the core enzyme for targeted screening, the dual-antibody thermal initiation technology, and the optimized buffer system, it demonstrates outstanding 3' end mismatch recognition ability. It can precisely distinguish miRNAs within the same family that differ by only one base pair, with a non-specific recognition rate lower than 5%.
Full instrument compatibility: It is equipped with a specially formulated ROX passive reference dye, eliminating the need to adjust the concentration according to the instrument model. It is widely applicable to various mainstream qPCR platforms.
Easy and efficient operation: It comes with universal U6 reference primers and universal reverse primers for humans, rats, and mice. Users only need to design specific forward primers themselves to easily conduct experiments.
| Cat. No. | Component | Specification | Quantity |
| B22011A | 2 × miRNA Unimodal SYBR qPCR Master Mix | 1 mL | 1 |
| B22021A | mQ Primer R (10 μM) | 40 μL | 1 |
| B22031A | U6 Forward Primer (10 μM) | 40 μL | 1 |
| B22041A | U6 Reverse Primer (10 μM) | 60 μL | 1 |
Note: The 2 × Master Mix contains SYBR Green I. Prepare and store the reagent away from light. The sequence of mQ Primer R is: AGTGCAGGGTCCGAGGTATT. U6 reference primers are applicable to human, rat and mouse samples. For other species, reference gene primers need to be designed and synthesized separately.
Store at -30 to -15℃ in a dark place and transport at ≤ 0℃. After thawing, the Master Mix can be stored stably at 2 to 8℃ in a dark environment for 6 months.
This product is applicable for miRNA expression profiling analysis, detection of miRNA expression levels in tissues/cells, and analysis of miRNA differential expression among different samples, etc.
Why did the experimental group show no amplification signal at all?
Possible reasons include: Poor quality of the RNA template: Check if the RNA is degraded. It is recommended to use agarose gel electrophoresis to detect the integrity of the RNA. Insufficient template dosage: It is recommended to add 0.5–1 μg of total RNA. The expression abundance of different miRNAs varies, and low-abundance miRNAs may require an increase in the template dosage. Mismatch between the reverse transcription product and the qPCR reagents: miRNA qPCR kits usually need to be used in conjunction with the same brand of reverse transcription kits because the reverse transcription primers are designed with specific PCR primer binding sequences, and the primer systems from different manufacturers may not be compatible. The target miRNA is not expressed in this sample: A positive control (such as U6 or a known highly expressed miRNA) can be set up to confirm the normality of the system.
Abnormal amplification curve (plateau phase decline, poor repeatability of re-coring)?
Possible causes and solutions: Excessive cDNA template concentration: It is recommended to dilute the cDNA appropriately (5-10 times) before conducting qPCR. Inappropriate primer concentration: It is suggested that the final concentration of the miRNA-specific forward primer should be approximately 200 nM. Inconsistent reaction system preparation: It is recommended to use pre-mixed solutions to reduce sampling errors. After adding the reagents, mix well and briefly centrifuge.
Did the melting curve show two peaks or a broad peak?
Possible causes and solutions: Non-specific amplification: Try raising the extension temperature from 60℃ to 65℃. Primer dimer formation: Appropriately reduce the primer concentration and pay attention to the concentration ratio of the upstream and downstream primers. Inappropriate primer design: It is recommended to redesign the specific forward primer to ensure its specific recognition of the target miRNA sequence. Wait for centrifugation.
How to improve the detection specificity of miRNA qPCR?
The sequences of members within the miRNA family are highly similar, sometimes differing by only 1-2 bases. Specific detection is a key challenge in miRNA qPCR. The core enzyme of this product has been selectively screened and possesses excellent 3' end mismatch recognition ability. Combined with the high-sealing-rate dual-species antibody thermal initiation technology, it can effectively distinguish miRNA members with highly similar sequences within the same family. It is recommended to cover the 3' end difference region of miRNA as much as possible when designing the forward primer to further improve specificity.
How to choose the appropriate ROX Reference Dye?
This product has been pre-mixed with a special formula of ROX Passive Reference Dye, suitable for various qPCR platforms. Different qPCR instruments have different requirements for ROX: Instruments requiring high ROX: ABI 7900HT, StepOnePlus, 7500, etc. Instruments requiring low ROX: ABI 7500 Fast, ViiA 7, etc. Instruments not requiring ROX: Bio-Rad CFX series, Roche LightCycler series, etc. This product has pre-mixed the ROX concentration compatible with various instruments, eliminating the need to adjust it manually according to different instruments.
What should we do if the Ct value of U6 internal control is too different from the target miRNA?
U6 is a snRNA, with a length of approximately 100 nt. Its expression abundance is usually much higher than that of most miRNAs, so the significant difference in Ct values is a normal phenomenon. However, it should be noted that: U6 is not a true miRNA. Its length (about 100 nt) is exactly at the edge of the retention range of the conventional RNA extraction column. If the short-chain RNA enrichment step is used, U6 may be partially lost during the extraction process, resulting in the loss of reference significance for the Ct values. In very unconventional samples such as serum, plasma, and exosomes, the content of U6 is unstable and varies greatly among individuals. It is recommended to select a more suitable reference based on the sample type (such as miR-16, miR-103, etc.).
How are the primers for miRNA qPCR designed?
This kit provides a universal reverse primer (mQ Primer R), with the sequence AGTGCAGGGTCCGAGGTATT. Users only need to design their own specific forward primers. The forward primers should be designed based on the mature sequence of the target miRNA. It is recommended that the Tm value be around 55–60℃. The length of the amplification product is usually about 80 bp. If using the stem-loop method, the reverse transcription primer can be designed by adding 6 bases to the miRNA sequence on the stem-loop sequence. Alternatively, primer design software can be used for assistance.
Can this kit be used for TaqMan probe-based qPCR?
This product is a pre-mix solution specifically designed for the SYBR Green conjugated fluorescence method. It has not been optimized for the probe method and is only suitable for qPCR using SYBR Green dye. If you need to use the probe method, please select the corresponding probe method-specific kit.
