miRNA fluorescent quantitative PCR detection kit
This kit adopts the principle of SYBR Green I chimeric fluorescence method for miRNA fluorescence quantitative detection. This kit contains all reagents for miRNA fluorescence quantitative detection, including 2 × MiRNA qPCR Mix and Reverse primer. two × MiRNA qPCR Mix (including Sybr Green) is a new generation of pre mixed form fluorescent quantitative PCR detection reagent developed specifically for miRNA quantitative detection. The DNA polymerase uses an antibody modified hot start form, combined with a special Buffer system, to improve reaction specificity, sensitivity, and accurate quantification over a wider range.
Component specifications
two × MiRNA qPCR Mix (With SYBR Green) 1.25mL
ten μ M Reverse prime 55 μ L
Storage: -20 ℃, protected from light, valid for 6 months.
Molecular biology experimental level water (without nuclease).
The qPCR upstream primer corresponding to the miRNA to be detected.
Forward Primer design principles:
Follow the most common principles of primer design.
Replacing U with T based on mature miRNA sequences is the most fundamental and simplest design method.
The Tm value of the downstream primer provided in the kit is 65 ℃, and the Tm value of the upstream primer should be designed to be around 65 ℃ as much as possible.
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.
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.
Thoroughly dissolve and mix before use. two × MiRNA qPCR Mix can be stored at 4 ℃ for short-term use to avoid repeated freezing and thawing. Store the Reverse Primer at -20 ℃ every time it is used up.
The addition amount of miRNA first strand cDNA should not exceed 1/10 of the real time PCR volume.
For special detection systems, high content of cDNA templates can easily lead to non-specific amplification, and cDNA should be appropriately diluted (10 or 100 times) based on the abundance of the detected miRNA.
This product contains the fluorescent dye Sybr Green I. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.
two × MiRNA qPCR Mix does not contain reference dye ROX. The customer will decide whether to add ROX reference dye according to the technical guidance of the qPCR instrument to eliminate signal background and correct fluorescence signal errors between wells.
This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit (ALH189).
Melt 2 x miRNA qPC at room temperature
