miRNA qPCR Detection Primer Set
I. Overview
1 Introduction
MicroRNAs (miRNAs) are a class of non-coding small RNAs of approximately 22 nucleotides that are widely found in eukaryotes. miRNAs have different expression patterns in different stages of development and in different tissues, indicating that they play a major regulatory role in development and differentiation. To date, miRNA detection methods mainly include molecular hybridization methods such as Northern Blot, which have low sensitivity, long time consumption, and large amount of RNA. The miRNA Q-PCR etection kit uses the internationally recognized standard for nucleic acid detection, Real-Time PCR, to detect miRNAs with fast, specific, and sensitive properties.
2, the principle of experiment
EzOmicsTM miRNA qPCR Detection Primer Set contains miRNA-specific reverse transcription primers, as well as qPCR positive and negative primer sets. A miRNA-specific reverse transcription primer in which a stem-and-loop structure is structurally transcribes a mature miRNA. Reuse of highly specific forward and reverse primers allows miRNA quantification to be sensitive and highly specific.
EzOmicsTM miRNA qPCR Detection Primer Set provides a highly sensitive detection method that detects up to 5 copies of a target miRNA with a dynamic range of 7 orders of magnitude. The template for the reaction can be total RNA or purified small RNA. Wait.
3, product content
name | Introduction | the amount |
RT Primer | Â miRNA Reverse Transcription Primer | 2 nmol |
Forward Primer | miRNA Forward Primer | 2 nmol |
Revers Primer | Universal Reverse Primer | 2 nmol |
Second, the experimental preparation
1. The customer needs to provide experimental reagent one-step or two-step qPCR mix, reverse transcriptase, RNase inhibitor, DEPC-H2O, etc.
2. Consumables are prepared without RNase, DNase tips, centrifuge tubes, etc.
3. Primer preparation primers are in the form of dry powder. Centrifuge first, then add 200 μL DEPC-H2O to each tube and dilute to 10 μM. Mix well and store at -20oC.
4. Instrument preparation centrifuge, real-time fluorescence quantitative PCR instrument, etc.
Third, the experimental steps
1. Two-step amplification first miRNAs are reverse transcribed miRNA cDNA, followed by qPCR quantification
1.1 RT Primer Mix Configuration
EzOmicsTM miRNA qPCR Detection Primer Set can detect up to 50 miRNA molecules simultaneously. RT Primer Mix (200 nM) is pre-configured prior to reverse transcription. If you need to use 5S rRNA or U6 snRNA as an internal reference control, you need to configure the Reverse Primer of the Random Primer or the internal reference as the reverse transcription primer.
1.2 Reverse transcription system
| Reagent | volume | Final concentration |
| RNA template | 0.2ng~1μg | According to experimental needs |
| RT Primer Mix | 5 μl | 20nM |
| 10×RT Buffer | 5 μl | 1× |
| RNase inhibitor | See reagent supplier instructions | |
| dNTP (2.5mM) | 4 μl | 200nM |
| Reverse transcriptase | See reagent supplier instructions | |
| DEPC-H2O | Make up to 50μl |
1.3 After the reverse transcription reaction is completed, store the miRNA cDNA at -20 °C (long-term storage, please store at -80 °C).
1.4 miRNA real-time quantitative PCR reaction system
1.5 Real-time quantitative PCR reaction procedure
2. One-step amplification:
Put the two processes of miRNA reverse transcription and qPCR quantification in one tube
2.1 Establishment of one-step miRNA quantification system
2.2 Real-time quantitative PCR reaction procedure
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