RT-PCR method for quantitatively detecting miRNA

A RT-PCR, quantitative detection technology, applied in the field of biomedicine, can solve problems to be improved and improved, and achieve the effects of simple operation, improved sensitivity and accuracy, and excellent simplicity

Active Publication Date: 2015-12-23
苟德明 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the S-Poly(T) method, the Poly(A) tailing and reverse transcription of miRNA are two independent reactions, so the S-Poly(T) technology needs to be improved in terms of ease of operation and reverse transcription efficiency and improve

Method used

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  • RT-PCR method for quantitatively detecting miRNA
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  • RT-PCR method for quantitatively detecting miRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1, S-Poly(T)Plus method for detection of circulating miRNA

[0059] The S-Poly(T)Plus method for detecting circulating miRNA is as follows: Figure 13 shown, including the following steps:

[0060] (1), extraction of serum total RNA

[0061] In this embodiment, the S / PmiRsol method is used to extract serum total RNA, and the specific steps are:

[0062] 1) Add 0.1pM nematode miRNAcel-miR-54 as an internal reference to 1mL RNAiso-Plus (TaKaRa) in advance, add 100μL serum, pipette and mix, let stand at room temperature for 5min; add 200μL chloroform, tightly cap the centrifuge tube, and shake vigorously for 20s ;Stand at room temperature for 5min;

[0063] 2) Centrifuge at 12,000g at 4°C for 15min; take out the centrifuge tube carefully, and the homogenate is divided into three layers, namely: a colorless supernatant (containing miRNA), a white protein layer in the middle, and a colored lower organic phase ;Pipe 500μL supernatant and transfer to another new ...

Embodiment 2

[0096] Example 2, S-Poly(T)Plus method for detecting miRNA in cells

[0097] (1) Extraction of total RNA in cells

[0098] In this embodiment, the specific steps for extracting total cellular RNA are:

[0099] 1) Add 10 6 For individual 293A cells, pipette and mix well, and let stand at room temperature for 5 minutes; add 200 μL of chloroform, tightly cap the centrifuge tube, shake vigorously for 20 seconds; let stand at room temperature for 5 minutes;

[0100] 2) Centrifuge at 12,000g at 4°C for 15min; take out the centrifuge tube carefully, and the homogenate is divided into three layers, namely: a colorless supernatant (containing miRNA), a white protein layer in the middle, and a colored lower organic phase ;Pipe 500μL supernatant and transfer to another new 1.5mL centrifuge tube;

[0101] 3) Add an equal volume of isopropanol (500 μL) to the supernatant, mix well by inverting up and down, and let stand at -20°C or -80°C for at least 10 minutes;

[0102] 4) Centrifug...

Embodiment 3

[0129] Example 3, S-Poly(T)Plus method detects the linear gradient range of miRNA in cells

[0130] This example analyzes the linear gradient range of the S-Poly(T)Plus method for detecting miRNA in cells. The human 293 cell RNA was diluted 5 times (2.5ng~0.8pg), and then six miRNAs (miR-92a-3p, miR-16-5p, miR-27b-3p, miR-210-3p, miR -103a-3p and miR-126-3p) and two internal reference snoRNAs (SNORD44 and SNORD7). From Figure 5 It can be seen from the figure that the linear correlation coefficient R of S-Poly(T)Plus detection of different miRNAs 2 (0.9933~0.9991) are greater than the value (0.9745~0.9968) of corresponding miRNA detected by S-Poly(T) method. Therefore, the S-Poly(T)Plus method for detecting cellular miRNA has a good linear relationship and a wide dynamic range.

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Abstract

The invention relates to an RT-PCR method for quantitatively detecting miRNA. The method is characterized in that Poly (A) tail adding and reverse transcription of miRNA are performed in the same reaction system, and S-Poly (T) primers are used to perform the reverse transcription of miRNA. The method has the advantages that in an S-Poly (T) Plus method, the Poly (A) tail adding and reverse transcription of miRNA are simultaneously performed in the same reaction system, simple operation is achieved, time is shortened, time for synthesizing cDNA is at least reduced by 0.5 hour, and high reverse transcription efficiency is achieved; an S/P miRsol RNA method and the S-Poly (T) Plus method are combined to build a technical system which is quite sensitive, efficient, simple, fast and cheap and capable of achieving extraction and detection; the technical system is especially suitable for detecting miRNA in biological fluid samples with low miRNA abundance.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a RT-PCR method for quantitatively detecting miRNA. Background technique [0002] microRNA (miRNA) is a kind of non-coding small RNA with a length of about 22 nucleotides, which widely exists in eukaryotic organisms such as animals, plants, and nematodes. The main function of miRNA is to inhibit gene expression by binding to the 3'-untranslated region (3'-UTR) of mRNA, degrade target mRNA or prevent its translation, thereby regulating gene expression at the post-transcriptional level. miRNA is widely involved in cell differentiation, proliferation, apoptosis, individual growth and development, and organ formation. miRNAs are precisely regulated in organisms, and their expression has strict spatiotemporal specificity. Studies have shown that the expression of miRNA is closely related to the occurrence of many cancers or other diseases, and miRNA can exist in a very stable form in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/113
Inventor 苟德明康康张利敏牛燕琴王志伟吴伊可张小英
Owner 苟德明
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