CN108929914A - A pancreatic cancer marker and its detection method - Google Patents

Abstract

The invention provides a pancreatic cancer marker, which is characterized by comprising two or more of the following detectable microRNA maturates stably existing in serum/plasma: miR-212, miR-21, miR-193b, miR-200c and miR-181 d; the invention also provides a detection method of the pancreatic cancer marker, which comprises the following steps: preparing a gene chip, extracting a pancreatic cancer marker gene miRNA of a sample to be detected, carrying out reverse transcription and amplification, hybridizing the gene chip, washing the gene chip, and analyzing results; the invention utilizes the gene chip to simultaneously detect two or more indexes influencing the pancreatic cancer occurrence and development, the obtained comprehensive analysis result has high accuracy, the detection efficiency is improved, and the detection cost is reduced.

Description

A pancreatic cancer marker and its detection method

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a pancreatic cancer marker and a detection method thereof.

Background technique

Pancreatic cancer is one of the common malignant tumors. In recent years, the incidence has been on the rise, and there is a lack of effective diagnostic methods and treatment measures, resulting in late detection of pancreatic cancer patients, poor prognosis, and high mortality. Regular screening of ordinary and high-risk groups is one of the effective measures to improve the early diagnosis rate and detect diseases in time.

In fact, the occurrence and development of pancreatic cancer involves changes in the expression of multiple genes. If the cancer is diagnosed at the gene level in the early stage of pancreatic cancer, and the expression changes of important related genes are detected, it will be beneficial to the early detection and early treatment of cancer, and improve the quality of life. patient survival rate.

The prototype of gene chip (genechip) (also known as DNA chip, biochip) was proposed in the mid-1980s. The sequencing principle of the gene chip is the method of hybridization sequencing, that is, the method of determining the nucleic acid sequence by hybridizing with a set of nucleic acid probes of known sequence, and the probe of the target nucleotide with known sequence is immobilized on the surface of a substrate. When the fluorescently labeled nucleic acid sequence TATGCAATCTAG in the solution is complementary to the nucleic acid probe at the corresponding position on the gene chip, a set of completely complementary probe sequences is obtained by determining the position of the probe with the strongest fluorescence intensity. This recombines the sequence of the target nucleic acid. As a high-throughput and advanced molecular biology technology, gene chip can be applied to the detection of gene expression changes in diseases. It is highly sensitive and accurate, fast and convenient, and can detect multiple gene expression changes at the same time, etc. advantage. At present, the gene chips for detecting pancreatic cancer can only detect a single indicator, and there is no report on the use of gene chips to simultaneously detect multiple indicators that affect the occurrence and development of pancreatic cancer.

Contents of the invention

The technical problem to be solved by the present invention is to provide a pancreatic cancer marker and a detection method thereof, which can simultaneously detect multiple indicators by using a gene chip, improve detection efficiency and accuracy, and reduce detection cost.

In order to solve the above-mentioned technical problems, embodiments of the present invention provide a pancreatic cancer marker, which is characterized in that it includes two or more of the following mature microRNAs that are stable in serum/plasma and detectable: miR-212, miR-21, miR-193b, miR-200c and miR-181d; wherein,

The miR-212 is hsa-miR-212-5p, and its sequence is: ACCUUGGCUCUAGACUGCUUACU;

The miR-21 is hsa-miR-21-5p, and its sequence is: UAGCUUAUCAGACUGAUGUUGA;

The miR-193b is hsa-miR-193-5p, and its sequence is: CGGGGUUUUUGAGGGCGAGAUGA;

The miR-200c is hsa-miR-200c-5p, and its sequence is: CGUCUUACCCAGCAGUGUUUGG;

The miR-181d is hsa-miR-181d-5p, and its sequence is: AACAUUCAUUGUUGUCGGUGGG.

The present invention also provides a method for detecting a pancreatic cancer marker according to claim 1, characterized in that it comprises the following steps:

S1. Prepare a gene chip. The gene chip includes a solid phase carrier and a sample to be tested fixed on the solid phase carrier. The solid phase carrier adopts an aldylated glass slide;

The gene chip is divided into a quality control part and a marker detection part, and the quality control part includes a negative control and a positive control; wherein, the negative control includes a blank group, a spotting solution group, and an Arabidopsis group, and the positive control includes an internal reference U6, yeast gene sequence 1, yeast gene sequence 2; the marker detection part includes hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p Oligo probe;

S2. Extracting pancreatic cancer marker gene miRNA from the sample to be tested: two or more of miR-212, miR-21, miR-193b, miR-200c and miR-181d;

S3, performing reverse transcription and amplification on the miRNA in the serum/plasma extracted in step S2, and then performing fluorescent labeling;

S4, gene chip hybridization;

S5, gene chip washing;

S6. Result analysis: the scanner scans the chip to detect the result of the hybridization reaction.

Further, the specific preparation method of the aldehydelated glass slide in the step S1 includes the following steps:

a. Use a glass slide as the substrate, clean the substrate, and shake it on a shaker at 60 rpm for 2 h. The cleaning solution is water, ethanol, and NaOH;

b. Take out the slide, wash it with ddH2O for 3 min, 4 times in total, and dry it with N2;

c. Soak the slide in Piranha solution for 30 min to make the slide show hydroxylation, and repeat step b; wherein, the Piranha solution includes H2SO4 with a concentration of 98% and H2O2 with a concentration of 30%, and the amount of both The ratio is 7:3;

d. Immerse the slide in 10 mM APTES prepared with absolute ethanol for 30 min, wash with absolute ethanol at 60 rpm for 1 h on a shaker, and blow dry with N2; wherein, the APTES is 3-ethylamino-tris Ethoxysilane;

e. Bake in an oven at 100-110°C for 1 h to fully combine APTES with the glass slide;

f. Soak in 10 mM terephthalaldehyde in acetone solution for 30 min, repeat step 2, and store in the dark at room temperature.

Further, the method for extracting miRNA in the step S2 includes the following steps: take 10 ml of blood sample and add 1ml TRIZOl, let it stand on ice for 10 min, the reagent is Invitrogen TRIzol; shake 200 ul of chloroform for 15 s, stand at 4°C for 2 min, 12000 g/15 min, carefully absorb about 500 ul of the middle layer; mix 500 ul isopropanol evenly, let stand at 4°C for 10 min, 12000 g/15 min; gently wash with 1 ml 75% ethanol, 7500 g/5 min, 2 times; dry at 55-65°C, dissolve 20-30 ulDEPC in water, promote dissolution at 65°C; colorimetric, A260/A280 is between 1.8 and 2.0, take 500 ng samples for agarose gel electrophoresis to verify the quality of product extraction .

Further, the kit used for reverse transcription in step S3 is Thermo Fisher Scientific’s RevertAid First Strand cDNA Synthesis Kit, and the following reagents were added in sequence to 200 ul enzyme-free EP tubes, and the whole process was performed on ice; Total RNA 0.1 ng-5 ug; hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p, hsa-miR-181d-5p stem-loop 1 ul; DEPC Make up water to 12 ul; 5X Reaction Buffer 4 ul; 20 U/μL RiboLock RNase Inhibitor 1 ul; 10 mM dNTP 2ul; 200 U/μL RevertAid M-MuLV RT 1 ul; Total volume 20 ul; In the 7500 PCR instrument, perform reverse transcription according to the program of 42°C for 60 min and 70°C for 5 min; add the following components in turn to the 200 ul EP tube: cDNA 5 ul; DEPC water 2 ul; 10X Reaction Buffer 5 ul; 25mmol mgcl2 4 ul ; 0.5 µl each of 100mM dATP, dCTP, and dGTP; 0.25 µl each of 100 mM dTTP, aa-dUTP; 1 ul each of upstream and downstream primers for miR-212, miR-21, miR-193b, miR-200c, miR-181d, and U6; Taqman 1 ul; centrifuge and mix well, and amplify in Roch 480 PCR instrument according to the following procedures: 95°C for 5 min; 95°C for 15 s; 60°C for 30 s; 72°C for 15 s; min; 4°C forever.

Further, the gene chip hybridization in the step S4 specifically includes the following process:

(1) Put the processed slides into the pre-hybridization solution at 42°C for 1 h;

(2) Rinse with ddH2O for 1 min, twice;

(3) Immerse in isopropanol for 1 min to ensure complete removal of SDS and dry by centrifugation;

(4) Denature the hybridization solution containing the sample cDNA at 95°C for 5 minutes, centrifuge for 2 minutes, return to room temperature and add the sample;

(5) Wash the coverslip with isopropanol, dry it, and place it flat on the hybridization box;

(6) Spot the hybridization solution onto the center of the coverslip without air bubbles;

(7) Gently cover the slide with the array face down on the coverslip, so that the hybridization solution fully covers the entire array, then turn the slide over and put it in the hybridization box;

(8) Add 10 ul 3X SSC to the grooves at both ends of the hybridization box, quickly cover the hybridization box lid, tighten the screws to avoid liquid immersion, and gently place it in a water bath at 42°C for overnight hybridization. The whole process is protected from light. .

Further, the step S5 of washing the gene chip specifically includes the following process: immerse the hybridized glass slide in the glass slide tank filled with the first washing solution, and the surface of the glass slide sample is facing down; remove the cover glass, and put the glass slide in the Shake in washing solution 1 for 2-3 times, then transfer to the slide tank filled with the second washing solution, shake the slide tank gently for 2 minutes, move the slides into the slide tank filled with the third washing solution and shake gently for 2 minutes , and then centrifuge to dry the slides; the whole process is protected from light; wherein, the first washing solution is a mixture of 2 × SSC and 0.1% SDS, the second washing solution is 1 × SSC, and the third washing solution is 0.2×SSC.

Further, the Oligo probes of hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p use DNA Synthesized by a synthesizer and modified by amination, then precipitated and purified with isopropanol, and each clone of the gene chip was placed on the substrate in a pin-point manner with a precise manipulator; then hydrated to make the probe The distribution is uniform; the amino group of the probe and the aldehyde group on the surface of the substrate undergo a Schiff base reaction to fix the probe; the spotting solution is the crystal core gene spotting solution; after the spotting is completed, the slide is placed for a week for ultraviolet cross-linking; finally For sealing, the glass slide is immersed in a 1-methyl-2-pyrrolidone solution of succinic anhydride, wherein the 1-methyl-2-pyrrolidone solution of succinic anhydride includes 1-methyl-2-pyrrolidone, succinic anhydride, Sodium borate; shake on a shaker in a fume hood for 20 min; take out the slides, rinse with ddH2O for 3 min, a total of 4 times, dry with N2, and store in the dark at room temperature.

Further, the Arabidopsis group in the negative control, the internal reference U6 in the positive control, the yeast gene sequence 1, and the yeast gene sequence 2 were all obtained by PCR amplification.

Further, the primer sequence of U6 in the step S1 is:

Upstream primer: CTCGCTTCGGCAGCACA;

Downstream primer: AACGCTTCACGAATTTGCGT;

The yeast gene sequence 1, its primer sequence is:

Upstream primer: AAGTTCCCTG CCGCCATCTT;

Downstream primer: CTCAATTGGA CAAATAATGC;

The yeast gene sequence 2, its primer sequence is:

Upstream primer: GTCCATGTAG GGTCTCTGGT;

Downstream primer: CTCAATTGGA CAAATAATGC;

The hsa-miR-212-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTAAG;

Upstream primer: ACCTTGGCTCTAGACTG;

Downstream primer: GTGCAGGGTCCGAGGT;

The hsa-miR-21-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA;

Upstream primer: TAGCTTATCAGACTGA;

Downstream primer: GTGCAGGGTCCGAGGT;

The hsa-miR-193b-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCATCT;

Upstream primer: CGGGGTTTTGAGGGCG;

Downstream primer: GTGCAGGGTCCGAGGT;

The hsa-miR-200c-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAACC;

Upstream primer: CGTCTTACCCCAGCAGT;

Downstream primer: GTGCAGGGTCCGAGGT;

The hsa-miR-181d-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCACCC;

Upstream primer: AACATTCATTGTTGTC;

Downstream primer: GTGCAGGGTCCGAGGT.

The beneficial effects of the above-mentioned technical solution of the present invention are as follows: the present invention utilizes the gene chip to simultaneously detect two or more indicators that affect the occurrence and development of pancreatic cancer, and the obtained comprehensive analysis results have high accuracy, improve the detection efficiency, and reduce the detection cost.

Description of drawings

Fig. 1 is the structural representation of gene chip among the present invention;

Fig. 2 is the detection flowchart of a kind of pancreatic cancer marker detection method in the present invention;

Fig. 3 is a diagram of the detection results of the gene chip in a method for detecting pancreatic cancer markers in the present invention.

Description of reference signs: 1. Blank group; 2. Spotting solution group; 3. Arabidopsis group; 4. Internal reference U6 group; 5. Yeast gene fragment 1 group; 6. Yeast gene fragment 2 group; 7. miR- 212 groups; 8, miR-21 group; 9, miR-193b group; 10, miR-200c group; 11, miR-181d group.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will describe in detail with reference to the drawings and specific embodiments.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", " The orientation or positional relationship indicated by "front", "rear", etc. is based on the orientation or positional relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation, are constructed and operate in a particular orientation and therefore are not to be construed as limiting the invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and should not be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that unless otherwise specified and limited, the terms "installation", "connection" and "connection" should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection. Connected, or integrally connected; it may be mechanically connected or electrically connected; it may be directly connected or indirectly connected through an intermediary, and it may be the internal communication of two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention in specific situations.

A pancreatic cancer marker, including two or more of the following mature microRNAs that are stable and detectable in serum/plasma: miR-212, miR-21, miR-193b, miR-200c and miR-181d; where,

The miR-212 is hsa-miR-212-5p, and its sequence is: ACCUUGGCUCUAGACUGCUUACU.

The miR-21 is hsa-miR-21-5p, and its sequence is: UAGCUUAUCAGACUGAUGUUGA.

The miR-193b is hsa-miR-193-5p, and its sequence is: CGGGGUUUUUGAGGGCGAGAUGA.

The miR-200c is hsa-miR-200c-5p, and its sequence is: CGUCUUACCCAGCAGUGUUUGG.

The miR-181d is hsa-miR-181d-5p, and its sequence is: AACAUUCAUUGUUGUCGGUGGG.

The present invention also provides a method for detecting a pancreatic cancer marker according to claim 1, comprising the following steps:

S1. Prepare a gene chip. The gene chip includes a solid-phase carrier and a sample to be tested fixed on the solid-phase carrier. The solid-phase carrier is an aldylated glass slide.

The gene chip is divided into a quality control part and a marker detection part, and the quality control part includes a negative control and a positive control; wherein, the negative control includes a blank group, a spotting solution group, and an Arabidopsis group, and the positive control includes an internal reference U6, yeast gene sequence 1, yeast gene sequence 2; the marker detection part includes hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p Oligo probe.

Wherein, the concrete preparation method of the glass slide of aldylation comprises the following steps:

a. Use a glass slide as the substrate, clean the substrate, and shake it on a shaker at 60 rpm for 2 h. The cleaning solution is water, ethanol, and NaOH;

b. Take out the slide, wash it with ddH2O for 3 min, 4 times in total, and dry it with N2;

c. Soak the slide in Piranha solution for 30 min to make the slide show hydroxylation, and repeat step b; wherein, the Piranha solution includes H2SO4 with a concentration of 98% and H2O2 with a concentration of 30%, and the amount of both The ratio is 7:3;

d. Immerse the slide in 10 mM APTES prepared with absolute ethanol for 30 min, wash with absolute ethanol at 60 rpm for 1 h on a shaker, and blow dry with N2; wherein, the APTES is 3-ethylamino-tris Ethoxysilane;

e. Bake in an oven at 100-110°C for 1 h to fully combine APTES with the glass slide;

f. Soak in 10 mM terephthalaldehyde in acetone solution for 30 min, repeat step 2, and store in the dark at room temperature.

Wherein, the Oligo probes of hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p are synthesized by DNA Synthesized by instrument, and carried out amination modification, and then precipitated and purified with isopropanol, and each clone of the gene chip was placed on the substrate in a pin-point manner with a precise manipulator; then hydrated to distribute the probes Uniform; the amino group of the probe and the aldehyde group on the surface of the substrate undergo a Schiff base reaction to fix the probe; the spotting solution is the crystal core gene spotting solution; after the spotting is completed, the slide is placed for a week for ultraviolet cross-linking; finally Sealed, immerse the slide in 1-methyl-2-pyrrolidone solution of succinic anhydride (reaction system: 1-methyl-2-pyrrolidone, succinic anhydride, sodium borate), and shake on the shaker in the fume hood for 20 min; take out Slides were rinsed with ddH2O for 3 min, 4 times in total, dried with N2, and stored in the dark at room temperature.

Wherein, the spotting liquid of the spotting group in the negative control is crystal core gene spotting liquid; the Arabidopsis group in the negative control, the internal reference U6 in the positive control, the yeast gene sequence 1, and the yeast gene sequence 2 all passed obtained by PCR amplification.

所述拟南芥保守区域序列为：ATGTTTCCTT CTCTAGATAC CAATGGCTAT GACCTCTTTGATCCCTTCATTCCCCATCAA ACAACCATGT TTCCTTCTTT CATTACTCAC ATTCAAAGCCCTAATTCTCACCATCACTAC TCTTCGCCTT CTTTTCCTTT CTCTTCCGATTTTCTTGAGA GTTTTGATGA ATCCTTCTTGATAAACCAAT TCTTGTTACAGCAGCAAGAT GTAGCAGCAA ATGTTGTTGA ATCTCCTTGG AAATTTTGCAAGAAGCTTGA GCTTAAGAAG AAGAATGAGA AGTGTGTTGA TGGAAGCACCTCACAAGAGG TTCAATGGAGAAGGACGGTC AAAAAAAGGG ACAGGCATG。

The internal reference U6 sequence in the positive control is:

ATGAGTCTTCGGAAGCAAACCCCTAGTGACTTCTTAAAGCAAAATCATCGGACGACCAGTTGTGGTAAAATTAAATTCTGGAGTGGATTATCGAGGGGTCCTGGCTTGCCTGGATGGCTACATGAATAGCCCTGGAGCAGACAGAAGAATATGTAAATGGACAACTGAAGAATAAGTATGGGGATGCATTTGATCCGAGGAAACAATGTGTTCAGTACATCAGGA.

The yeast gene sequence 1 sequence in the positive control is:

AAGTTCCCTGCCGCCATCTTGGGGCCCTGACATCACCAGGCACGTGGGTCCCGGCGGCCCCCGCGGGGCTCTAGTATATGAGATACTGGAATAATGTGTTTTTGTGTCAGGATTGTGG AAACAAACCCAATTATAAAAATGAACACATCCTTTTAAAAATTAACTTAATGTTTATTCAACAGGGTTGC.

The yeast gene sequence 2 sequence in the positive control is:

GTCCATGTAGGGTCCTGGTTTTATTTCTGCAGTTTCTTCGAAAGAGGAACAGGCCTGCTTTTAGTTAAGGTTAGTGTTTTATTTGTTATATAGTAACACACTGAAAGGATAATTTTGTTCAAAAATTTACTAAAAGATCTGGAAGGTGGTTGCTATGACAGATAATCAACTCAATTGGACAAATAATGC.

The Arabidopsis group in the negative control, the internal reference U6, the yeast gene sequence 1, and the yeast gene sequence 2 in the positive control were all obtained by PCR amplification.

The U6, its primer sequence is:

Upstream primer: CTCGCTTCGGCAGCACA

Downstream primer: AACGCTTCACGAATTTGCGT

The yeast gene sequence 1, its primer sequence is:

Upstream primer: AAGTTCCCTG CCGCCATCTT

Downstream primer: CTCAATTGGA CAAATAATGC

The yeast gene sequence 2, its primer sequence is:

Upstream primer: GTCCATGTAGGGTCTCTGGT

Downstream primer: CTCAATTGGA CAAATAATGC

The hsa-miR-212-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTAAG

Upstream primer: ACCTTGGCTCTAGACTG

Downstream primer: GTGCAGGGTCCGAGGT

The hsa-miR-21-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA

Upstream primer: TAGCTTATCAGACTGA

Downstream primer: GTGCAGGGTCCGAGGT

The hsa-miR-193b-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCATCT

Upstream primer: CGGGGTTTTGAGGGCG

Downstream primer: GTGCAGGGTCCGAGGT

The hsa-miR-200c-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAACC

Upstream primer: CGTCTTACCCCAGCAGT

Downstream primer: GTGCAGGGTCCGAGGT

The hsa-miR-181d-5p, its stem-loop and primer sequences are:

Stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCACCC

Upstream primer: AACATTCATTGTTGTC

Downstream primer: GTGCAGGGTCCGAGGT

The marker detection part is the Oligo probe of hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p, sequence They are:

The hsa-miR-212-5p sequence is: ACCTTGGCTCTAGACTGCTTACT.

The hsa-miR-21-5p sequence is: TAGCTTTATCAGACTGATGTTGA.

The hsa-miR-193b-5p sequence is: CGGGGTTTTGAGGGCGAGATGA.

The hsa-miR-200c-5p sequence is: CGTCTTACCCCAGCAGTGTTTGG.

The hsa-miR-181d-5p sequence is: AACATTCATTGTTGTCGGTGGG.

S2. Extract the pancreatic cancer marker gene miRNA of the sample to be tested: two or more of miR-212, miR-21, miR-193b, miR-200c and miR-181d; wherein, the miRNA extraction method includes The following steps: Take 10 ml blood sample and add 1 ml TRIZOl, let it stand on ice for 10 min, the reagent is Invitrogen TRIzol; shake 200 ul chloroform for 15 s, stand at 4°C for 2 min, 12000 g/15 min, carefully absorb the middle layer for about 500 ul; mix well with 500 ul isopropanol, let stand at 4°C for 10 min, 12000 g/15 min; gently wash with 1 ml 75% ethanol, 7500 g/5 min, twice; dry at 55-65°C, 20- Dissolve 30 ul of DEPC in water, promote dissolution at 65°C; colorimetric, A260/A280 is between 1.8 and 2.0, take 500 ng of sample for agarose gel electrophoresis to test the quality of sample extraction.

S3. Extract the miRNA in the serum/plasma in step S2, perform reverse transcription and amplification, and then perform fluorescent labeling. In order to detect a variety of miRNAs that may exist in serum, it is first necessary to extract miRNAs, reverse transcribe and amplify the extracted RNAs with the above primers, and then perform fluorescent labeling to hybridize with the target genes on the gene chip.

The reverse transcription steps are:

The kit used for reverse transcription was Thermo Fisher Scientific’s RevertAid First Strand cDNASynthesis Kit. The following reagents were added in sequence to 200 ul enzyme-free EP tubes, and the whole process was performed on ice.

Total RNA 0.1ng-5ug

hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p, hsa-miR-181d-5p stem-loop 1ul

DEPC water made up to 12ul

5X Reaction Buffer 4 ul

RiboLock RNase Inhibitor (20U/μL) 1 ul

10mM dNTP 2ul

RevertAid M-MuLV RT (200 U/μL) 1ul

Total volume 20ul

Centrifuge and mix well, and perform reverse transcription in an ABI 7500 PCR instrument according to the program of 42°C for 60 minutes and 70°C for 5 minutes.

Add the following ingredients in sequence to the 200ul EP tube:

cDNA 5ul

DEPC water 2ul

10X Reaction Buffer 5ul

25mmol mgcl2 4ul

dATP, dCTP, dGTP (100mM) 0.5µl each

dTTP, aa-dUTP (100mM) 0.25µl each

miR-212, miR-21, miR-193b, miR-200c, miR-181d, U6 upstream and downstream primers 1ul each

Taqman 1ul

Centrifuge and mix well, and amplify according to the following procedures in a Roch 480 PCR instrument:

95℃ 5min

95°C 15s

60℃ 30s

72°C 15s

40 cycles

72℃ 5min

4°C forever.

S4. Gene chip hybridization; gene chip hybridization specifically includes the following processes:

(1) Put the processed slides into the pre-hybridization solution at 42°C for 1 h;

(2) Rinse with ddH2O for 1 min, twice;

(3) Immerse in isopropanol for 1 min to ensure complete removal of SDS and dry by centrifugation;

(4) Denature the hybridization solution containing the sample cDNA at 95°C for 5 minutes, centrifuge for 2 minutes, return to room temperature and add the sample;

(5) Wash the coverslip with isopropanol, dry it, and place it flat on the hybridization box;

(6) Spot the hybridization solution onto the center of the coverslip without air bubbles;

(7) Gently cover the slide with the array face down on the coverslip, so that the hybridization solution fully covers the entire array, then turn the slide over and put it in the hybridization box;

(8) Add 10 ul 3X SSC to the grooves at both ends of the hybridization box, quickly cover the hybridization box lid, tighten the screws to avoid liquid immersion, and gently place it in a water bath at 42°C for overnight hybridization. The whole process is protected from light. .

S5, gene chip washing; gene chip washing specifically includes the following processes: immerse the hybridized glass slide in the glass slide tank filled with the first washing solution, and the glass slide sample is facing down; remove the cover glass, and place the glass slide in the washing After shaking in solution 1 for 2-3 times, move it into the slide tank filled with the second washing solution, shake the slide tank gently for 2 minutes, move the slide into the slide tank filled with the third washing solution and shake gently for 2 minutes, Then centrifuge and dry the slides; the whole process is protected from light; wherein, the first washing solution is a mixture of 2×SSC and 0.1% SDS, the second washing solution is 1×SSC, and the third washing solution is 0.2 ×SSC.

S6. Result analysis: the scanner scans the chip to detect the result of the hybridization reaction. Scan with ScanArray 3000 scanner, and use imagene to analyze hybridization results.

Analysis of negative control results:

A) The blank group is negative control 1. If there is no signal after hybridization, it indicates normal; if there is hybridization signal, the gene chip is contaminated.

B) The spotting solution is negative control 2. If there is no signal after hybridization, it indicates normal; if there is a hybridization signal, it indicates that the spotting solution is contaminated.

C) Arabidopsis is the negative control 3. If there is no signal after hybridization, it indicates normal; if there is hybridization signal, the gene chip is contaminated.

Negative controls 1, 2, and 3 have positive performance, and the gene chip cannot be used.

Analysis of positive control results

D), U6 is the positive control 1, if there is a signal after hybridization, it indicates normal; if there is no hybridization signal, it indicates that there is a problem with RNA extraction or reverse transcription.

E) Yeast gene sequence 1 is the positive control 2. The RNA is added to the serum in proportion during the process of extracting blood RNA, and then reverse-transcribed. If there is a hybridization signal, it indicates normal; if there is no signal, it indicates that RNA extraction or reverse transcription is defective. question.

F), Yeast gene sequence 2 is the positive control 3, and the DNA is added when the sample is subjected to PCR amplification and a fluorescent substrate is added. If there is a hybridization signal, it indicates normal; if there is no signal, it indicates that the labeling effect is not satisfactory.

Therefore, the quality control part of the scanning results: the positive control (internal reference U6, yeast gene sequence 1, yeast gene sequence 2) has fluorescence, and the negative control (blank group, spotting solution group, Arabidopsis group) has no fluorescence, which is normal, and the marker The substance detection part judges whether the detected pancreatic cancer markers are elevated according to the presence or absence of fluorescence. Show fluorescence as positive, not show fluorescence as negative.

As shown in Figure 3, it can be seen in Figure 3 that the part shown in group 1-3 is the negative control group without fluorescence, and the part shown in group 4-6 is the positive control group. Fluorescence indicates that the chip is qualified, and the parts shown in group 7-11 are respectively Pancreatic cancer markers miR-212, miR-21, miR-193b, miR-200c, and miR-181d were detected. Among them, groups 7, 8, and 11 had fluorescence, indicating that pancreatic cancer markers miR-212, miR21, and miR-181d were elevated; groups 9 and 10 had no fluorescence, and the expressions of miR-193b and miR-200c were normal. This gene chip simultaneously detected the negative control group, positive control group and pancreatic cancer markers miR-212, miR-21, miR-193b, miR-200c and miR-181d, among which, the pancreatic cancer markers miR-212, miR21, miR-181d is elevated, miR-193b and miR-200c are expressed normally, and multiple markers are detected simultaneously, which effectively improves the detection efficiency and the detection results are more accurate; combined with the analysis of the negative control group and the positive control group, the basic If the chip is contaminated or the detection process is invalid, the accuracy of the results will be comprehensively analyzed.

The above description is a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. A pancreatic cancer marker comprising two or more of the following microrna maturates stably present and detectable in serum/plasma: miR-212, miR-21, miR-193b, miR-200c and miR-181 d; wherein,

the miR-212 is hsa-miR-212-5p, and the sequence is as follows: ACCUUGGCUCUAGACUGCUUACU, respectively;

the miR-21 is hsa-miR-21-5p, and the sequence is as follows: uagcuuaucagucaguguuga;

the miR-193b is hsa-miR-193-5p, and the sequence is as follows: CGGGGUUUUGAGGGCGAGAUGA, respectively;

the miR-200c is hsa-miR-200c-5p, and the sequence is as follows: CGUCUUACCCAGCAGUGUUUGG, respectively;

the miR-181d is hsa-miR-181d-5p, and the sequence thereof is as follows: AACAUUCAUUGUUGUCGGUGGG are provided.

2. A method for detecting a pancreatic cancer marker according to claim 1, comprising the steps of:

s1, preparing gene chips, wherein each gene chip comprises a solid phase carrier and a sample to be detected fixed on the solid phase carrier, and the solid phase carrier adopts an aldehyde glass slide;

the gene chip is divided into a quality control part and a marker detection part, and the quality control part comprises a negative control and a positive control; wherein the negative control comprises a blank group, a sample solution group and an arabidopsis group, and the positive control comprises an internal reference U6, a yeast gene sequence 1 and a yeast gene sequence 2; the marker detection part comprises Oligo probes of hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5 p;

s2, extracting pancreatic cancer marker gene miRNA of the sample to be detected: two or more of miR-212, miR-21, miR-193b, miR-200c and miR-181 d;

s3, performing reverse transcription and amplification on miRNA in the serum/plasma extracted in the step S2, and then performing fluorescence labeling;

s4, gene chip hybridization;

s5, washing the gene chip;

s6, result analysis: the scanner scans the chip and detects the result of the hybridization reaction.

3. The method for detecting a pancreatic cancer marker according to claim 2, wherein the method for preparing the aldehyde-based slide glass in the step S1 specifically comprises the following steps:

a. cleaning a substrate by using a glass slide as the substrate, and shaking for 2 hours on a shaking table at 60rpm, wherein the cleaning solution is water, ethanol and NaOH;

b. taking out the slide, washing with ddH2O for 3 min for 4 times, and drying with N2;

c. b, soaking the glass slide in a Piranha solution for 30 min to ensure that the surface of the glass slide is hydroxylated, and repeating the step b; wherein the Piranha solution comprises 98% H2SO4 and 30% H2O2, and the ratio of the two is 7: 3;

d. immersing the slide in 10 mM APTES prepared by absolute ethyl alcohol for 30 min, cleaning the slide on a shaking table by using absolute ethyl alcohol at 60rpm for 1 h, and drying the slide by using N2; wherein the APTES is 3-ethylamino-triethoxysilane;

e. baking for 1 h in an oven at 100-110 ℃ to completely combine the APTES and the glass slide;

f. soaking in 10 mM acetone solution of terephthalaldehyde for 30 min, repeating step 2, and storing at room temperature in dark place.

4. The method for detecting a pancreatic cancer marker according to claim 2, wherein the extraction method of miRNA in step S2 comprises the following steps: adding 1ml of TRIZOL into 10 ml of blood sample, and standing on ice for 10 min, wherein the reagent is Invitrogen TRIZOL; shaking with 200ul chloroform for 15 s, standing at 4 deg.C for 2min, 12000 g/15min, and carefully sucking the middle layer for about 500 ul; uniformly mixing 500 ul isopropanol, standing at 4 ℃ for 10 min, and 12000 g/15 min; 1ml of 75% ethanol is used for gently washing, 7500 g/5 min, 2 times; oven drying at 55-65 deg.C, dissolving with 20-30 ul DEPC water, and promoting dissolution at 65 deg.C; and (4) carrying out color comparison, wherein A260/A280 is 1.8-2.0, and taking 500 ng of sample to carry out agarose gel electrophoresis to verify the extraction quality of the product.

5. The method for detecting pancreatic cancer markers as claimed in claim 2, wherein the reverse transcription in step S3 is performed using a Kit of RevertAid First Strand cDNAsynthesis Kit from Thermo Fisher Scientific, wherein the following reagents are sequentially added to 200ul of enzyme-free EP tube and are all performed on ice; total RNA0.1 ng-5 ug; hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p stem loop 1 ul; DEPC water to 12 ul; 5X Reaction Buffer 4 ul; 20U/. mu.LRiboLock RNase Inhibitor 1 ul; 10 mM dNTP 2 ul; 200U/uL RevertAid M-MuLV RT 1 ul; total volume 20 ul; centrifuging, mixing, and performing reverse transcription in ABI 7500 PCR instrument at 42 deg.C for 60min and 70 deg.C for 5 min; the following ingredients were added sequentially to a 200ul EP tube: 5 ul of cDNA; DEPC water 2 ul; 10 XRextraction Buffer 5 ul; 25mmol mgcl 24 ul; 0.5 μ l each of 100mM dATP, dCTP, dGTP; 100 mMdTTP, aa-dUTP each 0.25 μ l; 1ul of each of the upstream primer and the downstream primer of miR-212, miR-21, miR-193b, miR-200c, miR-181d and U6; taqman 1 ul; the mixture was centrifuged and amplified in a Roch 480PCR instrument according to the following protocol: 5min at 95 ℃; 15 s at 95 ℃;

30 s at 60 ℃; 15 s at 72 ℃; the above steps are circulated for 40 times; 5min at 72 ℃; 4 ℃ forever.

6. The method for detecting a pancreatic cancer marker according to claim 2, wherein the gene chip hybridization of step S4 specifically comprises the following steps:

(1) putting the treated glass slide into a prehybridization solution for prehybridization for 1 h at 42 ℃;

(2) washing with ddH2O for 1 min for 2 times;

(3) immersing the substrate in isopropanol for 1 min to ensure complete removal of SDS, and centrifugally drying;

(4) denaturing the hybridization solution containing the sample cDNA at 95 ℃ for 5min, centrifuging for 2min, recovering to room temperature, and adding the sample;

(5) cleaning the cover glass with isopropanol, drying, and then horizontally placing on a hybridization box;

(6) spotting the hybridization solution to the center of the cover glass without bubbles;

(7) lightly covering the glass cover with the array surface of the glass slide facing downwards to fully fill the whole array with the hybridization solution, and then turning over the glass slide and placing the glass slide into a hybridization box;

(8) respectively adding 10 ul 3 XSSC into grooves at two ends of a hybridization box, quickly covering a hybridization box cover, screwing a screw without liquid immersion, gently and horizontally placing the hybridization box cover in a water bath at 42 ℃ for hybridization overnight, and avoiding light in the whole process.

7. The method of detecting a pancreatic cancer marker according to claim 2,

the washing of the gene chip of the step S5 specifically comprises the following processes: immersing the hybridized slide into a slide groove containing a first washing solution, wherein the sample application surface of the slide faces downwards; removing the cover glass, shaking the glass slide in the washing liquid 1 for 2-3 times, then moving the glass slide into a glass slide groove containing the second washing liquid, gently shaking the glass slide groove for 2 minutes, moving the glass slide into a glass slide groove containing the third washing liquid, gently shaking the glass slide for 2 minutes, and then centrifugally spin-drying the glass slide; the whole process is carried out in a dark place; wherein the first washing solution is a mixed solution of 2 XSSC and 0.1% SDS, the second washing solution is 1 XSSC, and the third washing solution is 0.2 XSSC.

8. The method of claim 2, wherein Oligo probes hsa-miR-212-5p, hsa-miR-21-5p, hsa-miR-193b-5p, hsa-miR-200c-5p and hsa-miR-181d-5p are synthesized by a DNA synthesizer, aminated, precipitated with isopropanol, purified, and sequentially spotted on a substrate by a precise manipulator in a pin-spotting manner; then hydrating to ensure that the probes are uniformly distributed; the amino group of the probe and the aldehyde group on the surface of the substrate are subjected to Schiff base reaction so as to fix the probe; the sample solution is a crystal core gene sample solution; placing the slide for a week after sample application, and performing ultraviolet crosslinking; finally, sealing is carried out, and the glass slide is immersed into 1-methyl-2-pyrrolidone solution of succinic anhydride, wherein the 1-methyl-2-pyrrolidone solution of succinic anhydride comprises 1-methyl-2-pyrrolidone, succinic anhydride and sodium borate; shaking on shaking table in fume hood for 20 min; the slides were removed, washed with ddH2O for 3 min 4 times, dried with N2 and stored at room temperature in the dark.

9. The method of claim 2, wherein the Arabidopsis thaliana group in the negative control and the internal reference U6, the yeast gene sequence 1 and the yeast gene sequence 2 in the positive control are obtained by PCR amplification.

10. The method of claim 2, wherein the primer sequence of U6 of step S1 is:

an upstream primer: CTCGCTTCGGCAGCACA, respectively;

a downstream primer: AACGCTTCACGAATTTGCGT, respectively;

the yeast gene sequence 1 has a primer sequence as follows:

an upstream primer: AAGTTCCCTG CCGCCATCTT, respectively;

a downstream primer: CTCAATTGGA CAAATAATGC, respectively;

the yeast gene sequence 2 has a primer sequence as follows:

an upstream primer: GTCCATGTAG GGTCTCTGGT, respectively;

a downstream primer: CTCAATTGGA CAAATAATGC, respectively;

the stem loop and the primer sequence of the hsa-miR-212-5p are as follows:

stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTAAG, respectively;

an upstream primer: ACCTTGGCTCTAGACTG, respectively;

a downstream primer: GTGCAGGGTCCGAGGT, respectively;

the stem loop and the primer sequence of the hsa-miR-21-5p are as follows:

stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA, respectively;

an upstream primer: TAGCTTATCAGACTGA, respectively;

a downstream primer: GTGCAGGGTCCGAGGT, respectively;

the stem loop and the primer sequence of the hsa-miR-193b-5p are as follows:

stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCATCT, respectively;

an upstream primer: CGGGGTTTTGAGGGCG, respectively;

a downstream primer: GTGCAGGGTCCGAGGT, respectively;

the stem loop and the primer sequence of the hsa-miR-200c-5p are as follows:

stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAACC, respectively;

an upstream primer: CGTCTTACCCAGCAGT, respectively;

a downstream primer: GTGCAGGGTCCGAGGT, respectively;

the stem loop and the primer sequence of the hsa-miR-181d-5p are as follows:

stem-loop sequence: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCACCC, respectively;

an upstream primer: AACATTCATTGTTGTC, respectively;

a downstream primer: GTGCAGGGTCCGAGGT are provided.