Product content:
| M665794 | Component | 125 T | Storage | | M665794A | 2×miRNA qPCR Mixture (ROX) | 2×750 μL | -20℃. Avoid freeze/thaw cycle
| | M665794B | Reverse Primer, 10 μM | 60 μL | -20℃. Avoid freeze/thaw cycle
| | M665794C | ddH2O | 1.5 mL | -20℃. Avoid freeze/thaw cycle |
|
Product Introduction:
This kit uses the principle of SYBR Green I chimeric fluorescent dye method for miRNA fluorescence quantitative PCR detection. The kit includes 2 x miRNA qPCR Mixture and Reverse Primer required for detection. 2 x miRNA qPCR Mixture is a new generation pre mixed form of fluorescence quantitative PCR detection reagent specially developed for miRNA quantitative detection. The fluorescent dye SYBR Green I contained in it can bind to all double stranded DNA, making the product suitable for detecting different target sequences without the need to synthesize specific labeled probes. The GoldStar Taq DNA polymerase is a chemically modified and highly efficient thermal starter enzyme, coupled with a unique buffer system, which enhances reaction specificity, sensitivity, and enables accurate quantification of miRNA over a wider range. The 2x miRNA qPCR Mixture contains ROX dye and is suitable for fluorescence quantitative PCR instruments that require ROX as a calibration dye.
Note: This kit must be used in conjunction with the miRNA cDNA first strand synthesis kit.
Self prepared experimental materials: qPCR upstream primer. Forward Primer design principles:
1. Follow the most common principles of primer design.
2.Based on mature miRNA sequences, replacing U with T is the most basic and simplest design method.
3.The Tm value of the downstream primer provided in the reagent kit is 63.6 ℃, and the Tm value of the upstream primer should be designed to be around 63.6 ℃ as much as possible.
4. If the Tm value of the primer directly designed according to principle "2" is too low, several bases (preferably G or C bases) can be added to the 5 'end of the primer; One or several A bases can also be added at the 3 'end; Alternatively, both the 5 'and 3' ends can be modified simultaneously.
5.If the Tm value of a primer designed directly according to principle "2" is too high, several bases can be removed from the 5 'or 3' end of the primer. Notes:
1. Before using the reagent, please gently mix it upside down to avoid foaming, and use it after a brief centrifugation.
2. The amount of miRNA first strand cDNA added should not exceed 10% of the volume of Real time PCR.
3. For special detection systems, high content of cDNA templates can easily lead to non-specific amplification. Dilute cDNA appropriately (10 or 100 times dilution) based on the abundance of detected miRNAs.
4. The 2x miRNA qPCR Mixture in this product contains SYBR Green I and ROX dyes. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.
5. Avoid repeated freezing and thawing of this product. Repeated freezing and thawing may cause a decrease in product performance. This product can be stored at -20 ℃ for long-term storage. If frequent use is required in the short term, the 2xmiRNA qPCR Mixture can be stored at 2-8 ℃. However, the Reverse primer still needs to be stored at -20 ℃. Operation steps:
1. Melt 2 x miRNA qPCR Mixture and Reverse Primer at room temperature (10 μ M) .
2. When using, please gently mix the 2x miRNA qPCR Mixture upside down to avoid foaming, and use after brief centrifugation. If the reagent is not well mixed, its reaction performance will decrease.
3. Place the reagent on ice and prepare the reaction system according to the following table:
| reagent |
volume |
final concentration |
| 2×miRNA qPCR Mixture（ROX） |
10 μl |
1× |
| Forward primer（10 μM） |
0.4μl |
0.2 μM |
| Reverse primer（10 μM） |
0.4μl |
0.2 μM |
| MiRNA first strand cDNA |
X μl |
— |
| ddH2O |
up to 20 μl |
— | |
4. The reaction program is set as follows:
Attention!The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!
Note: 1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased. | 
