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Real-time fluorescence quantitative PCR detection kit for quantitative detection of GGPPS1 genes, detection method and application

A real-time fluorescence quantification and detection kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc. Easy to operate and simple effect

Inactive Publication Date: 2014-07-23
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the fact that there are few diagnostic indicators for clinical liver cancer detection and the accuracy is not particularly high, auxiliary molecular markers are needed to identify the occurrence and development of liver cancer. GGPPS1 plays an important role in the development of liver cirrhosis to liver cancer, and its expression also increases with The expression level increases with the progression of the disease

Method used

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  • Real-time fluorescence quantitative PCR detection kit for quantitative detection of GGPPS1 genes, detection method and application
  • Real-time fluorescence quantitative PCR detection kit for quantitative detection of GGPPS1 genes, detection method and application
  • Real-time fluorescence quantitative PCR detection kit for quantitative detection of GGPPS1 genes, detection method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Materials

[0046] (1) Reagents for total RNA extraction:

[0047] 1) Trizol extract 1ml;

[0048] 2) Chloroform 0.2ml;

[0049] 3) Isopropanol;

[0050] 4) 1% DEPC solution;

[0051] 5) 1ml of 75% cold ethanol (100% absolute ethanol: DEPC solution = 3:1);

[0052] (2) Reagents for reverse transcription of mRNA into cDNA template:

[0053] 1) RNase-free ultrapure water;

[0054] 2) Reverse transcription reaction solution 5×PrimeScript RT Master Mix 4μl (composed of PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture and Mg 2+ The composition of the reaction buffer); purchased from TAKARA company (Code No.RR036A)

[0055] (3) Reagents for Real Time PCR reaction:

[0056] 1) SYBR Premix Ex Taq II (TLi RNaseH Plus) (2×) 10 μl; purchased from TAKARA (Code No. RR820A)

[0057] 2) ROX Reference II (50×) 0.4 μl; purchased from TAKARA (Code No. RR820A)

[0058] 3) ultrapure water;

[0059] 4) GGPPS1 primer (10μM) upstream and downstrea...

Embodiment 2

[0071] Example 2 : To detect 2 kinds of liver tissue samples from 34 cases of liver cancer patients, respectively for the tissues:

[0072] (1) Extraction of sample RNA: take 30mg tissue specimen, suspend in 1ml Trizol, homogenize with a tissue homogenizer for about 1 minute, and let stand at room temperature for 5 minutes; add 0.2ml chloroform to the homogenate, shake for 15 seconds, Stand still for 2 minutes; centrifuge the standing solution at 4°C, 12,000 rpm for 10 minutes, and take the supernatant; add an equal volume of isopropanol to the supernatant, mix the liquid in the tube gently, and let stand at room temperature for 20 minutes to precipitate RNA; Then at 4°C, 12000rpm, centrifuge for 10 minutes, discard the supernatant; add 1ml of ice water pre-cooled to the precipitate and dilute to 75% ethanol solution with DEPC aqueous solution, gently wash the precipitate, and then at 4°C, 12000rpm, Centrifuge for 5 minutes, discard the supernatant, and dry; dissolve the dri...

Embodiment 3

[0079] Example 3 : Correlation analysis between GGPPS1 expression and clinicopathological features in liver cancer tissues

[0080] In order to detect the correlation between the expression of GGPPS1 in liver cancer tissues and clinicopathological features, immunohistochemical experiments were performed on the liver tissues of 70 clinical cases of liver cancer patients. The experimental results showed that the expression of GGPPS1 in liver cancer tissues had a great correlation with pathological factors ( Such as image 3 ), GGPPS1 expression was correlated with TNM stage (p=0.0059), tumor size (p=0.0041), vascular invasion (p=0.0009), early recurrence (p=0.0065), total recurrence rate (p=0.0444) and other factors Sex is statistically significant. Therefore, it can be concluded that the expression of GGPPS1 in the liver tissue of patients with liver cancer is higher, and it has a greater relationship with the clinical stage, blood vessel invasion, early recurrence an...

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Abstract

The invention discloses a fluorescence quantitative PCR kit for detecting GGPPS1 in a liver tissue of a liver cancer sample. The kit comprises the following ingredients: a specific primer and a fluorescence quantitative PCR reaction solution. The invention further discloses a using method of the fluorescence quantitative PCR kit for detecting the GGPPS1 in the liver tissue. By utilizing the kit, the expression of the GGPPS1 in the liver tissue can be fast and quantitatively detected, and the detection result can be used as a diagnosis standard of clinical liver cancer in early stage or advanced stage. The kit disclosed by the invention has the advantages of simplicity and convenience in operation, high speed, stable result, high sensitivity and strong specificity.

Description

technical field [0001] The present invention relates to the field of biological detection, more specifically, relates to the detection technology related to the change of mRNA expression in liver tissue of liver cancer. It is a kit that can accurately and quantitatively detect the expression level of GGPPS1 mRNA in samples by extracting total RNA from tissues or cells, and obtaining cDNA by reverse transcription of mRNA samples, combined with real-time fluorescent quantitative PCR detection technology. Background technique [0002] The GGPPS1 gene is the geranylgeranyl diphosphate synthase 1 (GGPPS1) gene. It plays a key role in the process of protein prenylation modification, and the substrate GGPP of GGPPS1 is an important precursor for the geranylation of small G proteins. Geranylation is one of the post-translational modifications of proteins, which generally occurs at the conserved cysteine ​​at the C-terminus of proteins. Geranylated proteins include Ras and Ras-rel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 薛斌李朝军刘佳黄旋
Owner NANJING UNIV
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