Method for detecting tumor microenvironment-associated antigen expression

A tumor microenvironment and antigen technology, which is applied in biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc. Strong reference effect

Inactive Publication Date: 2017-05-10
GUANGZHOU TAINUODI BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Tumor immunotherapy requires a comprehensive approach, and immunotherapy in the prior art usually only detects a few tumor-associated antigens, and cannot conduct a comprehensive and thorough analysis of the entire tumor microenvironment. Difficulty finding specific treatment options

Method used

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  • Method for detecting tumor microenvironment-associated antigen expression
  • Method for detecting tumor microenvironment-associated antigen expression
  • Method for detecting tumor microenvironment-associated antigen expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Sample RNA Extraction from Solid Tumor Tissue Samples

[0037] 1.1 Selection and preparation of tissue samples: surgically resected tumor tissue and benign tumor tissue used as a negative control, the margins were excised, and the intact tissue pieces in the middle were selected and stored in RNAlater at -80°C in the shortest possible time to prevent RNA degradation; Among them, RNAlater is a liquid, non-toxic tissue preservation reagent. It rapidly stabilizes tissues and preserves non-frozen cellular RNA in situ. After the tissue block is obtained, it is quickly soaked in RNAlater for preservation without causing RNA degradation, so that it is not necessary to process the sample immediately or freeze the sample in liquid nitrogen for later processing. It is a routine reagent in this field and can be obtained from commercial channels get.

[0038] 1.2 Take 30mg RNAlater to save the sample, add 1ml TRIzol, fully grind to lyse the tissue block, add chloroform...

Embodiment 2

[0043] Embodiment two: test sample RNA

[0044] The RNA concentration of the tissue sample was accurately measured with an ultra-micro spectrophotometer, wherein the model of the ultra-micro spectrophotometer used in this example was Nanodrop 2000. The quality analysis of RNA was carried out with a bioanalyzer, wherein the model of the bioanalyzer used in this example was Agilent 2100, and RNA samples with an OD value between 1.8 and 2.0 and a RIN value > 6 met the antigen expression profile detection standard , is a qualified RNA sample, which is used for the next experiment.

Embodiment 3

[0045] Example 3: Using the extracted sample RNA as a template and oligo dT as a primer to perform RNA reverse transcription to obtain tissue sample cDNA

[0046] Configure the cDNA acquisition reaction system shown in Table 1 on ice, mix the reaction system gently, centrifuge briefly, and then perform the procedures shown in Table 2 to obtain template cDNA.

[0047] Table 1. cDNA acquisition reaction system

[0048] components volume 10X RT Buffer (buffer) 2.0ul 25X dNTP Mix 0.8ul 10X oligo(dT) 2.0ul Reverse Transcriptase 1.0ul RNase Inhibitor 1.0ul sample RNA 1ug Nuclease-free H 2 O (nucleic acid-free pure water)

up to 20ul

[0049] Table 2. cDNA acquisition reaction program

[0050] step 1 step 2 step 3 step 4 temperature 25℃ 37℃ 85℃ 4℃ time 10min 120min 5min ∞

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Abstract

The invention discloses a method for detecting tumor microenvironment-associated antigen expression. The method comprises the following steps of (1) extracting sample RNAs from solid tumor tissue samples; (2) checking the sample RNAs; (3) taking the extracted and qualified sample RNAs as templates, and carrying out inverse transcription of the RNAs by employing oligo dT as a primer to obtain tissue sample cDNAs; and (4) designing a qPCR detection primer, amplifying the tissue sample cDNAs through qPCR amplification and collecting fluorescence signals. The method has the beneficial effects that an inventor selects multiple tumor microenvironment-associated antigens, and correspondingly designs multiple antigen primers; and the expression conditions of the antigens in the tissue samples are specifically detected through current main gene expression analysis method and a fluorescent quantitative PCR method. Through the method, the microenvironment-associated antigen expression values of tumor patients are obtained and the expression values have high reference when a doctor provides the patients with targeted therapeutic schedules.

Description

technical field [0001] The invention belongs to the technical field of antigen expression detection, and in particular relates to a method for detecting the expression of tumor microenvironment-related antigens. Background technique [0002] Recent studies have found that the microenvironment of various tumors, especially solid tumors, has a strong inhibitory effect on the body's anti-tumor immune response. These inhibitory mechanisms mainly include the following aspects: First, PD-L1 / PD-L2 expressed on the surface of tumor cells transmits inhibitory signals to PD-1 on the surface of T lymphocytes. Since PD-1 is an inhibitory receptor, the killing function of T lymphocytes on tumor cells is inhibited. Over the past year, the US FDA has successively approved anti-PD-1 antibodies to treat lung cancer, kidney cancer, and anti-PD-L1 antibodies to treat advanced bladder cancer, indicating that blocking the tumor microenvironment’s suppression of tumor immune responses is a very ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/136C12Q2600/158
Inventor 何有文廖化新栗世铀郑伟宏肖云鹏李军旗王茜婷袁晓辉
Owner GUANGZHOU TAINUODI BIOTECH
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