Primers, Taqman probe and kit for detection of STMN1 mRNA expression quantity

A technology of expression quantity and kit, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of inability to recognize the difference between cDNA and genome, affect the accuracy of test results, and is not conducive to finding conserved regions, etc. problem, to achieve the effect of avoiding false positive results, high accuracy and high degree of automation

Inactive Publication Date: 2016-03-16
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The SYBRgreen method is easy to operate, relatively cheap, and has relatively high sensitivity, so it is favored. Many scientific researches use the SYBRgreen method. However, because SYBRgreen dyes bind to double-stranded DNA and are not specific, they are also prone to false positives. Positive situation; the Taqman method has the characteristics of high sensitivity and good specificity, so this method is generally used in clinical practice, but because the temperature required for probe binding is high, the fragment length of the probe is generally relatively long, which is not It is beneficial to find the conserved regions of shorter fragments of all sorted sequences. In addition, the increase of probe length also reduces the efficiency of probe binding and affects the accuracy of detection results.
In addition, the conventional Taqman probe method cannot identify the difference between cDNA and genome, and cannot avoid false positives caused by the adulterated genome during RNA extraction

Method used

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  • Primers, Taqman probe and kit for detection of STMN1 mRNA expression quantity
  • Primers, Taqman probe and kit for detection of STMN1 mRNA expression quantity
  • Primers, Taqman probe and kit for detection of STMN1 mRNA expression quantity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Primer screening and kit composition and use

[0056] 1. STMN1 amplification primers

[0057] Oligonucleotide primer pair STMN1-F (SEQ ID NO:1) and STMN1-R (SEQ ID NO:2) for robust, reproducible STMN1 gene expression by PCR using non-reverse-transcribed cDNA from paraffin-embedded tissue Quantitative. The oligonucleotide primer pair STMN1-F (SEQ ID NO: 1) and STMN1-R (SEQ ID NO: 2) have extremely high specificity and repeatability, and have high amplification efficiency.

[0058] STMN1-F: 5'-ACCATGGCTTCTTCTGATATCCAG-3', SEQ ID NO: 1;

[0059] STMN1-R: 5'-ATTCTTTTGACCGAGGGCTG-3', SEQ ID NO: 2;

[0060] STMN1 Taqman-MGB probe: 5'-FAM / AACTGGAGAAGCGTGC / MGB-3', SEQ ID NO:3.

[0061] The length of the amplified product of STMN1-F (SEQ ID NO: 1) and STMN1-R (SEQ ID NO: 2) using oligonucleotide primers is 83 base pairs. The amplified product corresponds to the amplified part of exons 2 and 3 of STMN1 cDNA. The oligonucleotide primers have significantly higher s...

Embodiment 2

[0096] Example 2: Extraction and reverse transcription of clinical sample RNA

[0097] 1. RNA extraction and reverse transcription of mRNA from paraffin tissue samples

[0098] In this example, RNA is extracted from the cancer tissue and paracancerous tissue cells of the paraffin tissue sample of lung cancer patient A, and the integrity of the total RNA in the tissue is identified by electrophoresis, and quantified by an ultraviolet spectrophotometer. Ningbo Youcheng Paraffin Embedded Tissue (FFPE) Total RNA Extraction Kit (Cat. NO: KR001A20) was used, and the operation was performed according to the instructions. The details are as follows:

[0099] 1. Cut the paraffin sample into 5-10 μm thick slices.

[0100] 2. Place 4-8 slices in a 1.5mL RNase-free centrifuge tube (prepared by yourself). Add 300 μL wax removal solution DB.

[0101] 3. Place the centrifuge tube at 80°C and incubate for 30 minutes, take it out every 10 minutes during the period, mix by hand or vortex bri...

Embodiment 3

[0145] Example 3: Detection of STMN1 expression in cancer tissues and paracancerous tissues of lung cancer patients by real-time fluorescent PCR

[0146] In this example, the expression level of STMN1 in the mRNA (cDNA) sample extracted in Example 2 was detected by using the kit provided by the present invention, and it was detected by immunohistochemical method as a comparison.

[0147] The reverse-transcribed cDNA was used as a template to carry out fluorescent quantitative PCR, wherein the fluorescent quantitative PCR reaction system was:

[0148] PCR reaction solution:

[0149] MgCl 2 : 2.0~5.0mmol

[0150] dNTPs: 0.2~0.8mmol

[0151] Each primer: 0.1~1.0μmol

[0152] Each probe: 0.1~1.0μmol

[0153] FastAdvancedMasterMix: 3~6μL

[0154] Template: 5 μL

[0155] Total volume: 25 μL.

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Abstract

The invention discloses primers, a Taqman probe and a kit for detection of STMN1 mRNA expression quantity, wherein the primers have the nucleotide sequences shown in SEQ ID No:1-2, and the Taqman probe has the nucleotide sequence shown in SEQ ID NO:3; a 5' end of the probe is labeled with a fluorescent reporter group, and a 3' end of the probe is labeled with a quencher. The primers and the probe have high sensitivity and good specificity; the kit composed of the primers and the probe is easy and safe to operate, has the whole detection process requiring only 80 min, saves the time, has relatively high sensitivity and specificity on all of fresh frozen tissues, paraffin-embedded tissues, blood and other samples, and has wide application range; relative quantification adopts a double-standard-curve method, an error is relatively small, and a result is more accurate.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to primers, Taqman probes and kits for detecting the expression level of STMN1 mRNA. Background technique [0002] Microtubule instability protein (STMN1) is a highly conserved intracellular soluble phosphoprotein, which regulates the dynamic balance of the cellular microtubule system through phosphorylation and dephosphorylation at different stages of the cell cycle, controls the cell cycle, changes Biological behaviors such as cell proliferation and differentiation. Its regulatory effect on microtubules is an important guarantee for the formation of the spindle in mitosis, so it has also become a direct target of anti-microtubule drugs. [0003] Anti-microtubule drugs are a class of broad-spectrum chemotherapeutic drugs that affect the formation of spindles and inhibit cell mitosis by acting on cellular microtubules. Clinically, it is widely used in the treatment of ovari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/101
Inventor 胡利红夏琦吴志伟段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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