Pancreatic cancer drug tolerance detection method and kit

A detection kit and detection method technology, applied in the field of genetic engineering, can solve problems such as drug resistance, and achieve the effects of high specificity, low cost, and high prediction accuracy

Active Publication Date: 2017-08-11
NANJING BIOHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical problem that pancreatic cancer cells are prone to produce drug resistance to drug generatives in the prior art, and provides a detection method and kit for drug resistance of pancreatic cancer

Method used

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  • Pancreatic cancer drug tolerance detection method and kit
  • Pancreatic cancer drug tolerance detection method and kit
  • Pancreatic cancer drug tolerance detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Pancreatic cancer clinical drug Gemze drug-resistant cell line obtained

[0026] Pancreatic cancer cells PANC-1 and MIA PaCa-2 were screened for drug-resistant cell lines using gemcitabin (Gemzar), a standard chemotherapy drug for pancreatic cancer. Through drug dose-escalation screening method, PANC-1 and MIA PaCa-2 drug-resistant cell lines that can survive in 56.4 μM and 15.0 μM Gemzar respectively were obtained. The semi-lethal inhibitory concentration (IC50) analysis showed that the IC50 of the drug-resistant cell lines PANC-1-R and MIA PaCa-2-R was increased by 121 times and 6 times, respectively, compared with the drug-sensitive cell lines. The increase in the expression of related drug resistance proteins is a key factor for cancer to acquire drug resistance. For example, MRP1 and MDR1 are upregulated to varying degrees in the acquisition of drug resistance of most cancers. Therefore, we further detected the changes of PANC-1-R (PR) and MIA PaCa-2-R (MR) resist...

Embodiment 2

[0035] RNA Extraction from Pancreatic Cancer and Gemzeitan Drug-resistant Tissues

[0036] 1) Grind the tissue with liquid nitrogen, add 1mL Trizo to each sample, lyse, let it stand at room temperature for 5min, and transfer it to a 1.5mL centrifuge tube;

[0037] 2) Add 200 μL of chloroform, shake fully or turn it upside down several times, after the lysate turns pink, let stand at room temperature for 5 minutes;

[0038] 3) Centrifuge at 12,000g for 15min at 4°C, absorb the supernatant and put it into another 1.5ml centrifuge tube; add 500μL isopropanol, mix the liquid in the tube upside down, and let stand at room temperature for 10min;

[0039] 4) Centrifuge at 12,000g for 10min at 4°C, discard the supernatant;

[0040] 5) Add 1mL of 75% ethanol to slowly wash the white precipitate; centrifuge at 8,000g for 10min at 4°C, discard the supernatant;

[0041] 6) Dry in the air, add an appropriate amount of ultrapure water (RNase-free) to dissolve the precipitate, which can be...

Embodiment 3

[0043] Target miRNA detection in pancreatic cancer and Gemzeita drug-resistant samples

[0044] To detect miR-584, miR-153, miR-188, and miR-483 in the RNA extracted in Example 2, the primers used are as follows:

[0045]

[0046] 1. Reverse transcription PCR:

[0047] Reverse transcription PCR (RT-PCR)

[0048]

[0049] Reverse transcription program: Specific primers: 42°C for 15min, 85°C for 5sec, 4°C to terminate the reaction.

[0050] The cDNA used in the next step for fluorescent quantitative PCR amplification was obtained by reverse transcription PCR.

[0051] 2. Real-time fluorescent quantitative PCR:

[0052] Real-time fluorescence quantitative PCR (qRT-PCR) reaction system (20μL)

[0053]

[0054] qRT-PCR program: 95°C 30s pre-denaturation; 95°C 3s, 30s, 60°C 30s, 40 cycles. Internal reference for U6 miRNA. Use ABI 7500fast Real-Time PCR instrument and software for analysis. RNA relative expression = 2 -△△CT , ΔCT=target gene CT-internal reference CT ...

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Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a pancreatic cancer drug tolerance detection method and kit. Expression of miR-584, miR-153, miR-188 and miR-483 in a pancreatic cancer drug-tolerant tissue sample is remarkably higher than that of the same in a pancreatic cancer normal tissue sample, which can serve as a pancreatic cancer drug-tolerance screening marker to provide accurate clinical medication guidance for clinical patients. The pancreatic cancer drug tolerance miRNA detection kit takes combined use of miR-584, miR-153, miR-188 and miR-483 as a pancreatic cancer drug tolerance detection index and is based on fluorescent real-time quantitative PCR (polymerase chain reaction); the kit is quick and convenient in detection, high in detection sensitivity and specificity, low in cost, capable of meeting needs on detecting tolerance of most of pancreatic cancer drugs, extremely wide in application range and high in prediction accuracy through clinical verification.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a detection method and a kit for pancreatic cancer drug resistance. Background technique [0002] Non-coding RNA refers to the RNA that is transcribed by the genome but not translated into protein. Among them, microRNA (miRNA) is a kind of small molecule non-coding RNA with evolutionary conservation, which widely exists in animals, plants and pathogenic microorganisms. The miRNA gene is transcribed by RNA polymerase II or III to produce an original miRNA transcript (Primary miRNA) with a length of hundreds to thousands of nucleotides, which is then passed through two RNase III family enzymes Drosha and Dicer is processed in two steps to generate mature miRNAs with a length of about 22 nucleotides. After being assembled into the effector complex RISC (RNA-induced silencing complex), miRNA locates RISC to the target mRNA through base pairing, triggers translational repression or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/106C12Q2600/158C12Q2600/178
Inventor 贺小宏王延宾袁鹏王鑫张承武李林
Owner NANJING BIOHENG BIOTECH CO LTD
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