CN102154475A - Kit for detecting ERCC1 mRNA (Excision Repair Cross Complement Group 1 Messenger Ribonucleic Acid) expression by using fluorescence quantitative PCR (Polymerase Chain Reaction) technology - Google Patents

Abstract

The present invention relates to the field of biotechnology. A fluorescent quantitative PCR diagnostic kit for rapid detection of ERCC1 mRNA, the purpose is to provide a fast, simple, sensitive, and specific detection kit for ERCC1 mRNA and its application. The expression status of DNA repair genes may be relatively ideal for chemotherapy efficacy predictive factor. Nucleotide excision repair (NER) is closely related to platinum drug resistance, among which excision repair cross-complementation gene 1 (ERCC1) is an important factor involved in the repair process and leading to platinum drug resistance, with high sensitivity and specificity The specificity and sensitivity of the detection results are significantly improved in the detection of ERCC1 mRNA levels by fluorescent quantitative PCR. This kit provides a new, fast and simple genetic diagnosis technology for clinical malignant tumor patients whether to use platinum-based chemotherapy drugs.

Description

A kit for detecting the expression level of ERCC1 mRNA by fluorescent quantitative PCR technique

technical field

The invention relates to the field of biotechnology, in particular to a kit for detecting the expression of ERCC1 mRNA by means of fluorescent quantitative PCR technology.

Background technique

Since the discovery of cisplatin's anticancer activity in 1967, the application and research of platinum-based metal anticancer drugs have developed rapidly. Cisplatin has the characteristics of broad anti-cancer spectrum and strong effect. It is one of the most commonly used drugs in current chemotherapy. Platinum drugs combine with intracellular DNA to cause inter-strand cross-linking or intra-strand cross-linking, causing DNA damage. resulting in cell death. However, after cytotoxic chemotherapeutic drugs such as cisplatin damage the tumor, the DNA repair mechanism of tumor cells is activated, and tumor cells with high repair ability tend to be resistant to chemotherapy.

Studies suggest that the expression status of DNA repair genes can be used as a relatively ideal predictor of chemotherapy efficacy. Nucleotide excision repair (NER) is closely related to platinum drug resistance, and excision repair cross-complementation gene 1 (ERCC1) is an important factor involved in the repair process and leading to platinum drug resistance (van Duin M, de Wit J, Odijk H, et a1. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and amino acid homology with the yeast DNA repair gene RAD10. Cell. 1986; 44(6): 913-923). Recent studies have found that the expression level of ERCC1 gene is related to the incidence, biological characteristics and prognosis of breast cancer, liver cancer, esophageal cancer, gastric cancer, ovarian cancer and non-small cell lung cancer (McDaniel LD, Schultz RA. XPF/ERCC4 and ERCC1: their products and biological roles. Adv Exp Med Biol. 2008; 637: 65-82). The different expression of ERCC1 is closely related to the choice of treatment of non-small cell lung cancer, the application of drugs after surgery and the judgment of prognosis (Simon GR, Begum M, Bepler G. Setting the stage for tailored chemotherapy in the management of non-small cell lung cancer. Future Oncol. 2008 Feb;4(1):51-59). It has been reported in the literature that patients with high expression of ERCC1 after surgery for early non-small cell lung cancer have a significantly longer survival time and a better prognosis. It can be used as an independent predictor to guide the prognosis of non-small cell lung cancer; in patients with advanced non-small cell lung cancer receiving platinum-based chemotherapy, those with positive expression of ERCC1 have a worse prognosis than those with negative expression, and the disease-free survival of those with negative ERCC1 And the overall survival time was significantly longer than that of ERCC1-positive patients. In vivo and in vitro studies have further confirmed that the expression level of ERCC1 gene is positively correlated with the resistance of cells to platinum drugs, and the high expression of ERCC1 is one of the most important mechanisms leading to the resistance of tumor cells to platinum drugs (Martin L P, Hamilton TC, Schilder RJ. Platinum resistance: the role of DNA repair pathways. Clin Cancer Res. 2008;14(5):1291-1295).

The 273-amino acid protein encoded by ERCC1 does not have the function of nucleotide excision repair. Therefore, when studying the relationship between ERCC1 and chemotherapy efficacy, the quantitative detection of ERCC1 protein content by immunohistochemistry cannot reflect the function of ERCC1 gene, but the sensitivity and specificity can be used. The mRNA levels were detected by fluorescent quantitative PCR. At the same time, the pathological types and clinical stages of non-small cell lung cancer commonly used in clinical practice are not ideal for predicting the curative effect and survival of patients. A large number of studies hope to find some relevant molecular markers of non-small cell lung cancer in order to treat patients. and prognosis to provide more beneficial help. The expression of ERCC1 gene meets this requirement. It is not only helpful for the estimation of the prognosis of non-small cell lung cancer, but also more instructive for the selection of drugs for adjuvant chemotherapy after surgery.

At present, there is no literature report on the kit for detecting the expression of ERCC1 mRNA.

Contents of the invention

The purpose of the present invention is to provide a fast, simple, sensitive and specific kit for detecting the expression level of ERCC1 mRNA.

The kit of the present invention utilizes fluorescent quantitative PCR technology and includes ERCC1 gene primers, internal reference gene (β-actin) primers and Taqman fluorescent probes:

The upstream primer sequence of ERCC1 gene is: 5′-GGGAATTTGGCGACGTAATTC-3′ (SEQ ID NO: 1);

The downstream primer sequence of ERCC1 gene is: 5'-GCGGAGGCTGAGGAACAG-3' (SEQ ID NO: 2);

Taqman fluorescent probe: 6FAM 5'-CACAGGTGCTCTGGCCCAGCACATA-3'TAMRA (SEQ ID NO: 3);

The upstream primer sequence of β-actin gene is: 5'-TGAGCGCGGCTACAGCTT-3' (SEQ ID NO: 4);

The downstream primer sequence of β-actin gene is: 5'-TCCTTAATGTCACGCACGATTT-3' (SEQ ID NO: 5);

Taqman fluorescent probe: 6FAM 5'-ACCACCACGGCCGAGCGG-3'TAMRA (SEQ ID NO: 6).

The kit also includes reagents needed for fluorescent quantitative PCR.

The kit of the present invention specifically includes: first-strand cDNA synthesis reagents, negative controls and standards, PCR reaction solution, RNA extraction reagents and the like.

The first-strand cDNA synthesis reagents are:

25mmol/L MgCl2 _4ul ,

10× reverse transcriptase buffer 2ul,

10mmol/L dNTP 2ul,

RNase inhibitor 0.5ul,

Oligo(dT) ₁₅ 0.5ug,

AMV reverse transcriptase 15U,

DEPC water;

Wherein the negative control and positive control: deionized water is used as the negative control, and the sample containing ERCC1 total RNA is used as the positive control.

The PCR reaction solution includes: 10×PCR Premix, 0.25pmol/ul primers (ERCC1 gene primers and internal reference gene primers), 0.3pmol/ul fluorescent probes, 2.5-4.0mM Mgcl2, 2U Taq enzyme, 0.2 -0.4mM dNTPs, 0.2-1U UNG enzyme, 0.3-0.6mM dUTP, usually take 1-2ul template.

Use the kit of the present invention to detect ERCC1 mRNA expression: first obtain clinical cancer tissue samples, quickly extract tissue RNA, and perform reverse transcription PCR to synthesize the first-strand cDNA; Read out the CT value result in the analysis system. The Ct values of ERCC1 and β-actin genes were calculated respectively, and the difference between them was the ΔCt value. The results of fluorescent quantitative PCR were analyzed by software, and the sample data were standardized and calculated.

The advantages and effects of the test kit of the present invention are as follows:

(1) Sensitive: Fluorescent PCR detection technology is a technology that integrates PCR technology, fluorescent labeling technology, laser technology, and digital imaging technology, so its detection sensitivity is very high.

(2) Specificity: Using specific probes to identify quantitative molecules has high accuracy. At the same time, the target sequence is double-controlled by primers and probes, with good specificity and low false positives.

(3) Simple and safe: simple operation, safe, high degree of automation, anti-pollution. Amplification and detection can be detected in the same tube, no need to open the cap, and it is not easy to be polluted; at the same time, amplification and detection are completed in one step, no post-processing is required, and there is no need to worry about radioactive contamination.

(4) Fast: Fast, high-throughput, can be completed within 3-4 hours.

Description of drawings

Fig. 1A is a fluorescence curve diagram of ERCC1 standard substance detected by fluorescence real-time quantitative RT-PCR.

Fig. 1B is a standard curve obtained from the fluorescence curve graph of ERCC1 standard.

Fig. 2A is the fluorescence curve of the β-actin standard detected by fluorescence real-time quantitative RT-PCR.

Fig. 2B is a standard curve obtained from the fluorescence curve of the β-actin standard.

Detailed ways

Now, the present invention will be further described in conjunction with the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

Embodiment 1. Preparation of kit of the present invention

(1) The kit of the present invention consists of the following:

①Trizol: rapid extraction of cancer tissue RNA;

② First strand cDNA synthesis kit (RT-PCR);

③ Primers: including the target gene ERCC1, the internal reference gene GAPDH and fluorescent probes, as follows:

ERCC1 gene primer sequence:

Upstream: 5′-GGGAATTTGGCGACGTAATTC-3′;

Downstream: 5′-GCGGAGGCTGAGGAACAG-3′;

Taqman fluorescent probe: 6FAM 5’-CACAGGTGCTCTGGCCCAGCACATA-3’TAMRA

β-actin gene primer sequence:

Upstream: 5'-TGAGCGCGGCTACAGCTT-3';

Downstream: 5′-TCCTTAATGTCACGCACGATTT-3′;

Taqman fluorescent probe: 6FAM5'-ACCACCACGGCCGAGCGG-3'TAMRA.

The above primer sequences and probe sequences were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

④Negative control and positive control: use deionized water as negative control, and use ERCC1 total RNA sample as positive control.

⑤PCR reaction solution: 10×PCR Premix, 0.25pmol/ul primer, 0.3pmol/ul probe, 2.5-4.0mM Mg2+, 2U Taq enzyme, 0.2-0.4mM dNTPs, 0.2-1U UNG enzyme, 0.3-0.6mMdUTP, usually take 1-2ul template, the total reaction volume is usually 20ul (all the above concentrations refer to the final concentration)

⑥PCR amplification program setting: On the lightcycler2.0 instrument, it is usually 50°C for 10s, 95°C for 10min, then 95°C for 15 seconds and 60°C for 1min, and cycle 46 times.

Embodiment 2. detect the expression level of ERCC1 mRNA with the kit prepared in embodiment 1

Take the detection results of 30 cases of non-small cell lung cancer paraffin section specimens as an example.

Detection process:

Firstly, specific primers and fluorescent probes are designed according to the gene sequence. Obtain clinical cancer tissue samples, quickly extract tissue RNA, and perform RT-PCR to synthesize first-strand cDNA; configure PCR reaction solution for fluorescent quantitative PCR. Read out the CT value results in the LightCycler data analysis system. The Ct values of ERCC1 and β-actin genes were calculated respectively, and the difference between them was the ΔCt value. The results of fluorescent quantitative PCR were analyzed by software, and the sample data were standardized and calculated.

Specific steps are as follows:

①Extraction of total tissue RNA: extract total RNA from cancer and paracancerous tissues according to the method of RNA extraction and purification. The integrity of the extracted RNA was identified by agarose gel electrophoresis, the purity and concentration of the RNA were calculated by measuring the 260nm and 280nm optical density values with a UV spectrophotometer, and the extracted RNA was adjusted to the same concentration with 0.1% DEPC-treated water.

② cDNA synthesis by reverse transcription: Take 2ul of the above RNA solution and incubate at 70°C for 10min, then add 25mmol/L MgCl ₂ 4ul, 10× reverse transcriptase buffer 2ul, 10mmol/L dNTP 2ul, RNase inhibitor 0.5ul, Oligo( dT) ₁₅ 0.5ug, AMV reverse transcriptase 15U, add DEPC-treated water to a total volume of 20ul, incubate at 42°C for 15min, carry out reverse transcription reaction, and synthesize the first strand of cDNA. After the reaction, heat to 99°C for 5 minutes to inactivate the reverse transcriptase. Add 20ul sterile water, mix well and store in the refrigerator at -20°C.

④ Fluorescent quantitative PCR amplification: PCR reaction system is 20ul: 2Xpremix 10.0ul, ERCC1 primer concentration 0.2μmol/L, probe concentration 0.3μmol/L, cDNA 1.0ul, ultrapure water to make up. React on a lightcycler fluorescent quantitative PCR instrument: Amplification conditions: 95°C 10min pre-denaturation, 95°C 15s, 60°C 1min amplification for 40 cycles, the instrument automatically collects fluorescence signals.

⑤ Data collection, processing and analysis: Calculate the data obtained by real-time fluorescent quantitative PCR to obtain the relative expression of the target gene relative to the housekeeping gene (β-actin), and then perform statistical analysis, and the ratio is greater than 0.5 as high expression:

samples ERCC1 template β-actin template ERCC1/β-actin Non-small cell lung cancer 247347739 346698765 0.71 Non-small cell lung cancer 143267584 316876506 0.45 Non-small cell lung cancer 98644566 324579017 0.30 Non-small cell lung cancer 356576782 307556842 1.16 Non-small cell lung cancer 235795436 319458540 0.74 Non-small cell lung cancer 87654333 334561573 0.26 Non-small cell lung cancer 298564746 290645734 1.03 Non-small cell lung cancer 187598466 315787518 0.59 Non-small cell lung cancer 236764385 296451487 0.80 Non-small cell lung cancer 87570654 319653319 0.27 Non-small cell lung cancer 268403709 298546190 0.90

Non-small cell lung cancer 297557900 317650194 0.94 Non-small cell lung cancer 97954681 316406153 0.31 Non-small cell lung cancer 288569043 286487947 1.01 Non-small cell lung cancer 167598460 339839599 0.49 Non-small cell lung cancer 253764385 286631489 0.89 Non-small cell lung cancer 97850645 318654321 0.31 Non-small cell lung cancer 280421709 308546156 0.91 Non-small cell lung cancer 298157590 306671049 0.97 Non-small cell lung cancer 89649078 298565890 0.30 Non-small cell lung cancer 85470794 279537187 0.31 Non-small cell lung cancer 189448164 278976590 0.68 Non-small cell lung cancer 273894360 328506467 0.83 Non-small cell lung cancer 98646940 327517465 0.30 Non-small cell lung cancer 97850645 318654321 0.31 Non-small cell lung cancer 280421709 308546156 0.91 Non-small cell lung cancer 298157590 306671049 0.97 Non-small cell lung cancer 89649078 298565890 0.30 Non-small cell lung cancer 285407794 299537187 0.95 Non-small cell lung cancer 189456164 318977310 0.59

(2) Evaluation of the detection ability of the kit:

Preliminary experiments show that the sensitivity, specificity and sensitivity of this kit are more accurate than immunohistochemistry, and fully meet the practical requirements of current clinical diagnosis and treatment:

in:

① Specificity: 100%;

②Sensitivity: 95%;

③ Positive predictive value: the positive predictive value reaches 100%;

④Negative predictive value: The negative predictive value reaches 93%;

⑤Repeatability: The results of repeated experiments are consistent;

⑥Time-consuming: The detection time of a clinical specimen is about 4 hours, which is short.

The above experiments can show that the specificity and sensitivity of the detection results are significantly improved by using fluorescent quantitative PCR with high sensitivity and specificity to detect the level of ERCC1 mRNA. This kit provides a method for clinical tumor chemotherapy and prognosis. A new fast and easy genetic diagnosis technology.

Claims (2)

1. test kit that utilizes fluorescent quantitative PCR technique to detect ERCC1 mRNA expression amount comprises ERCC1 gene primer, internal control gene β-actin primer and Taqman fluorescent probe:

ERCC1 upstream region of gene primer sequence is: 5 '-GGGAATTTGGCGACGTAATTC-3 ';

ERCC1 gene downstream primer sequence is: 5 '-GCGGAGGCTGAGGAACAG-3 ';

Taqman fluorescent probe: 6FAM 5 '-CACAGGTGCTCTGGCCCAGCACATA-3 ' TAMRA;

β-actin upstream region of gene primer sequence is: 5 '-TGAGCGCGGCTACAGCTT-3 ';

β-actin gene downstream primer sequence is: 5 '-TCCTTAATGTCACGCACGATTT-3 ';

Taqman fluorescent probe: 6FAM 5 '-ACCACCACGGCCGAGCGG-3 ' TAMRA.

2. a kind of test kit that utilizes fluorescent quantitative PCR technique to detect ERCC1 mRNA expression amount according to claim 1, it is characterized in that this test kit specifically comprises: the first chain cDNA synthetic agent, negative control and positive control, PCR reaction solution and RNA extract reagent;

The first chain cDNA synthetic agent wherein is:

25mmol/L?MgCl ₂?4ul，

10 * reversed transcriptive enzyme damping fluid 2ul,

10mmol/L?dNTP?2ul，

RNA enzyme inhibitors 0.5ul,

Oligo(dT) ₁₅?0.5ug，

AMV reversed transcriptive enzyme 15U,

DEPC water;

Wherein negative control and positive control: with the negative contrast of deionized water, to contain the positive contrast of the total RNA sample of ERCC1;

PCR reaction solution wherein comprises: the UNG enzyme of the Taq enzyme of the fluorescent probe of the ERCC1 gene primer of 10 * PCR Premix, 0.25pmol/ul and internal control gene primer, 0.3pmol/ul, Mgcl2, the 2U of 2.5-4.0mM, dNTPs, the 0.2-1U of 0.2-0.4mM, 0.3-0.6mM dUTP, get the template of 1-2ul usually.

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