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Primer group for detecting gene expression of tumor metabolic pathway, PCR (Polymerase Chain Reaction) chip and application thereof

A primer set, tumor technology, applied in DNA/RNA fragments, combinatorial chemistry, organic compound library, etc., can solve the problems of high price and high cost, and achieve the effect of low cost, high accuracy and wide application prospects.

Active Publication Date: 2017-09-01
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it often only contains genes of a certain metabolic pathway. For example, the glucose metabolism PCR chip detects 84 key genes related to the regulation of glucose and glycogen metabolism and enzyme pathways, so it has certain limitations, and its price is relatively expensive, about 4800 Yuan / plate / sample, if a large number of samples and several channels are to be tested, the cost will be extremely high

Method used

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  • Primer group for detecting gene expression of tumor metabolic pathway, PCR (Polymerase Chain Reaction) chip and application thereof
  • Primer group for detecting gene expression of tumor metabolic pathway, PCR (Polymerase Chain Reaction) chip and application thereof
  • Primer group for detecting gene expression of tumor metabolic pathway, PCR (Polymerase Chain Reaction) chip and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Design of primers for PCR amplification of genes related to tumor metabolic pathways

[0055] 1. Through a large number of preliminary studies, the inventor team identified 168 genes related to tumor metabolic pathways, and their gene names and functional classifications are shown in Table 1.

[0056] Table 1 168 genes related to tumor metabolic pathways of the present invention and their functional classification

[0057]

[0058]

[0059] 2. Through a lot of research, the team of the present invention designed and screened specific amplification primers corresponding to 168 genes related to tumor metabolic pathways. The amplification efficiency of the primers is high, and the CG content of the primers is between 35% and 65%. The annealing temperature (Tm) is between 60-68°C, the length of the primers is 10-23bp, and the size of the amplified fragment is 100-200bp.

[0060] 3. Select β-actin, PRKG1, GAPDH, GUSB and HMBS as internal reference genes.

...

Embodiment 2

[0081] Example 2 Preparation of Metabolic PCR Chip

[0082] 1. The primer set designed in Example 1 was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0083] 2. Dissolve the synthesized primers in RNase-free water (RNase-free water) with a final concentration of 10 μM. Operate on ice, minus 20°C, and save for later use.

[0084] 3. Preparation of the PCR chip master plate: add primers (forward and reverse primers mixed) into the round-bottom 96-well plate according to the wells corresponding to the PCR chip gene primers, 200 μl / well.

[0085] Preparation of the PCR chip working plate: Use a high-throughput automatic pipetting system to dispense 80 μl of primers from the master plate into a round-bottom 96-well plate containing 160 μl of RNase-free water; further use a high-throughput automatic pipetting system to transfer the 96 10.5 μl of the primers in the well plate were dispensed into a PCR plate, and stored at minus 20°C for future use.

...

Embodiment 3

[0087] Example 3 Tumor Cell RNA Extraction and Reverse Transcription

[0088] 1. Cell culture: Lymphoma cell lines SU-DHL-2, Raji, Jeko-1, etc. and leukemia cell line MV411 were cultured according to the instructions.

[0089] 2. RNA extraction and reverse transcription

[0090] (1) Pretreatment: glassware was treated overnight with 0.1% DEPC water, and baked at 240°C for 4 hours. Plastic equipment was treated overnight with 0.1% DEPC water, 1.034×10Spa, sterilized under high pressure at 121°C for 20min, and dried.

[0091] (2) Collection of cells and extraction of total RNA:

[0092] a. Cell collection (5×10 6 ~10 7 ) Discard the medium, wash with PBS (DEPC water treatment), add 1ml Trizol to the cells, pipette several times, transfer the cell suspension to a 1.5ml eppendorf tube, and let stand at room temperature for 5min;

[0093] b. Add 0.2ml chloroform for every 1ml Trizol to the cell homogenate, shake vigorously for 15sec, place at room temperature for 2min, centrif...

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Abstract

The invention discloses a primer group for detecting the gene expression of a tumor metabolic pathway, a PCR (Polymerase Chain Reaction) chip and a detection method thereof. The primer group and the PCR chip comprise primer pairs which are used for respectively and specifically multiplying 168 genes relevant to the tumor metabolic pathway, wherein sequences of the primer pairs are as shown from SEQ ID NO.1 to SEQ ID NO.336 in sequence. By verification and screening of the primer specificity of the 168 genes of the tumor metabolic pathway and optimization of a PCR multiplication condition, fragments of all target genes can be simultaneously multiplied under the same multiplication condition. The PCR chip disclosed by the invention is high in repetitiveness, high in accuracy, easy to operate and low in cost; simple, quick and accurate measures are provided for finding expressions of abnormal genes of the tumor metabolic pathway and the relevance between the abnormal gene and a tumor phenotype; the primer group and the PCR chip have an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set for detecting gene expression of tumor metabolic pathways, a PCR chip and applications thereof. Background technique [0002] Compared with normal cells, the metabolic changes and abnormalities of tumor cells are another important feature of tumors, which are mutually causal with the occurrence and development of tumors. Even in the case of sufficient supply, the mitochondrial function defects and the changes in the tricarboxylic acid cycle metabolism of tumor cells lead to redox metabolism disorders, so that a large amount of glucose can be absorbed through the glycolysis pathway to generate energy to meet the needs of rapid growth. This is the famous Warburg effect. Changes in tumor metabolism have become one of the new tumor characteristics (Hallmarks of Cancer). Therefore, it is a major scientific issue in the field of tumor research to explore the key d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6886C12Q2600/118C40B40/06
Inventor 黄蓬刘晓霞胡寓旻卢文华姜伟业
Owner SUN YAT SEN UNIV CANCER CENT
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