miRNA Reagents
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Features
● High sensitivity: Target miRNA of total RNA of pg grade can be detected.
● Strong specificity: The completely upgraded system can better distinguish single base differences and perfectly distinguish different miRNA within family.
● Good versatility: Suitable for various types of fluorescence quantitative PCR instruments.
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Description
The kit adopts the principle of SYBR® Green I chimeric fluorescence to carry out miRNA fluorescence quantitative detection. The kit contains all reagents for quantitative miRNA fluorescence detection, including 2× miRcute Plus miRNA Premix, 50× ROX Reference Dye and Reverse Primer. Among them, 2× miRcute Plus miRNA Premix (With SYBR&ROX) is a new generation of pre-mixed fluorescent quantitative PCR detection reagent specially developed for miRNA quantitative detection. The DNA Polymerase uses chemically modified hot-start polymerase, cooperating with a special buffer system, it has better reaction specificity, higher sensitivity, and accurate quantification can be carried out in a wider range.
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Kit Contents
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Applications
Detect reverse transcribed cDNA of miRNA by fluorescence quantitative PCR. -
Required Reagents
● RNase-Free ddH2O
● Forward primer
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Storage Condition
The miRcute Plus miRNA qPCR Kit (SYBR Green) should be stored immediately upon receipt at -30~-15°C, protected from light. Thaw each components and mix thoroughly before use. If the 2× miRcute Plus miRNA Premix (SYBR&ROX) are thawed but not used, it is important to thoroughly mix them prior to refreezing (The layering of salts during the thawing process and subsequent crystallization during freezing will damage the enzyme and decrease product performance). This kit could be stored at -30~-15°C for 1 year. -
Important Notes
● This kit must be used together with miRcute Plus miRNA First-Strand cDNA Kit (4992786/4992909).
● The kit contains SYBR Green I. Store and prepare PCR reaction in dark condition.
● Pipet the reaction solution and aliquot with sterile tips and consumable items to avoid contamination.
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Experimental Example
Table 1. There are 10 kinds of 3p miRNA in the human let-7 family, with little or even single base difference among them. The 10 miRNA mimics were used as templates and different primers were used to amplify the 10 miRNA respectively by using the TIANGEN miRcute Plus miRNA qPCR Kit (SYBR Green). The results show that the specific primer only amplifys the specific template, and the non-specific recognition rate is less than 5%, indicating that TIANGEN miRcute Plus miRNA qPCR Kit (SYBR Green) has good specificity and can distinguish miRNA with single base difference.
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Compatible Real-time Instruments
● PRISM 7000/7300/7500/7700/7900HT Real-Time PCR System, Step one/Step one plus PCR System (Applied Biosystems)
● LightCycler (Roche)
● Mx3000P, Mx3005P and Mx 4000 (Stratagene)
● Mastercycler ep realplex (Eppendorf)
● Line-Gene (Bioer)
● Other Real-time instruments
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Forward Primer Design Principles
● Follow the most common design principles of primers.
● Based on mature miRNA sequence, change U to T to design forward prime.
● Tm value of Reverse Primer (10 µM) in the kit is about 65°C. Ensure that Tm value of forward prime is basically consistent with supplied Reverse Primer (10 µM).
● If Tm value of the designed forward prime is too low according to principle 2, it is recommended to add several nucleotides (G or C is optional) to 5'-terminal, or add one or several adenines to 3'-terminal. Optionally, modify both 5' terminal and 3' terminal at the same time.
● If Tm value of the designed forward prime is too high according to principle 2, remove several nucleotides from 5' or 3' terminal.
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PCR Program Outlined in the Following Table
● In general, a real-time PCR reaction can be set up as follows:
● To improve the detection specificity and detection rate of low abundance miRNA, the following procedure can be used:
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