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Method for detecting miRNA (micro Ribose Nucleic Acid) by improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction)

A technology of stem-loop primers and detection primers, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of high cost, inability to fully guarantee the specificity of cDNA sequences, and the inability to analyze the specificity of PCR reactions. High accuracy and precision, technology cost savings, good sensitivity results

Inactive Publication Date: 2014-06-18
DONGHUA UNIV
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Problems solved by technology

However, the cost of this method is high, and the Taqman probe only binds to part of the cDNA sequence of the reverse transcription product, and its specificity cannot be fully guaranteed, and the specificity of the PCR reaction cannot be analyzed through the melting curve

Method used

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  • Method for detecting miRNA (micro Ribose Nucleic Acid) by improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction)
  • Method for detecting miRNA (micro Ribose Nucleic Acid) by improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction)
  • Method for detecting miRNA (micro Ribose Nucleic Acid) by improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction)

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Embodiment 1

[0041] 1. Design of miRNA detection primers

[0042] (1) Stem-loop primers consist of two parts, a universal stem-loop sequence and a specific primer sequence complementary to the 3'end of the target miRNA; the optimized stem-loop primer increases the number of bases that complement the target miRNA from 7bp to 11bp.

[0043] (2) The downstream primers of qPCR are aimed at the universal stem-loop sequence, and the upstream primers are aimed at the 5'end of the target miRNA, and the 5'end has an 8bp tail. The primer design is as follows figure 2 Shown.

[0044] 2. Verify the specificity and sensitivity of primers

[0045] (1) Reverse transcription

[0046] SuperScript II Reverse Transcriptase Kit (Fermentas) was used for reverse transcription. The final reaction system 10μL includes: 6.25μL DNase I-treated total RNA, 0.5μL stem-loop primer (2μM), 0.5μL dNTP Mix (10mM). First, the reaction was carried out at 16°C for 15 minutes, and then the remaining system (2μL 5×RT buffer, 0.5μL Reve...

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Abstract

The invention relates to a method for detecting miRNA (micro Ribose Nucleic Acid) by an improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction). The method comprises the steps: (1), designing a miRNA detection primer, wherein a stem-loop primer comprises two parts of a general stem loop sequence and a specific primer sequence which is in complementary pairing with a 3' end of a target miRNA, a base number which is in complementary pairing with the target miRNA is 11bp, a downstream primer needle of qPCR is specific to a general stem loop sequence, an upstream primer needle is specific to a 5' end of the target miRNA, a GC tail is added in the 5' end of the upstream primer so that the annealing temperatures of the upstream primer and the downstream primer are close; (2), performing inverse transcription on the target miRNA into cDNA (complementary Desoxvribose Nucleic Acid); and (3), performing a quantitative PCR amplification and detecting by adopting an SYBR Green qPCR detection method. The method disclosed by the invention is good in specificity, and high in sensitivity; and one downstream primer is only synthesized, and an SYBR Green I fluorochrome method is adopted, and thus the cost is greatly saved.

Description

Technical field [0001] The invention belongs to the field of miRNA detection methods, and particularly relates to an improved stem-loop primer qRT-PCR method for miRNA detection. Background technique [0002] MicroRNAs (miRNAs) are a type of single-stranded non-coding RNA composed of approximately 22 nucleotides, which mainly regulate the expression of target genes at the post-transcriptional level through direct shearing of messenger RNA or indirect inhibition of translation. MicroRNAs have different expression patterns in different stages of individual development, such as cell differentiation, proliferation, apoptosis, etc. and in different tissues, indicating that they play an important regulatory role in development and differentiation. [0003] Traditional miRNA detection technologies such as northern hybridization and microarray have poor sensitivity to the amplification of low-abundance miRNAs. The qRT-PCR (Quantitative reverse transcription PCR) method is the most sensiti...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2525/207C12Q2525/301C12Q2563/107
Inventor 薛慧慧肖君华周宇荀李凯
Owner DONGHUA UNIV
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