CN101698886A - Liquid phase chip for detecting mRNA expression of gene associated with treatment effect of carboplatin and detection method - Google Patents

Abstract

This invention discloses a liquid phase chip for detecting mRNA expression level of a gene associated with treatment effect of carboplatin, which mainly comprises a microsphere coupled with an amido-modified support probe, support extension probes connecting the support probe and mRNA of the target gene, and amplification extension probes connecting the mRNA of the target gene and signal detection components, wherein each support extension probe comprises a specific sequence of which the 5' end can be combined with the mRNA of the corresponding target gene, a spacer arm sequence, and a sequence of which the 3' end can be complementarily paired with a specific sequence of a corresponding support probe; and each amplification extension probe comprises a specific sequence of which the 5' end can be combined with the mRNA of the corresponding target gene, a spacer arm sequence, and a sequence of which the 3' end can be complementarily paired with a labeled probe. The invention also discloses a method for detecting the mRNA expression level of the gene associated with treatment effect of the carboplatin by the liquid phase chip. The detection process does not require steps such as RNA extraction, reverse transcription, PCR and the like, and detection results are little influenced by the quality of RNA in a sample, and are ensured to be accurate; moreover, because of the liquid phase chip technology, multi-index parallel detection is realized, a plurality of reference genes can be arranged in once detection, and the detection results are more reliable.

Description

MRNA expression of gene associated with treatment effect of carboplatin detects liquid-phase chip and detection method

Technical field

The invention belongs to biology field, relate to medical science and biotechnology, the concrete mRNA expression of gene associated with treatment effect of carboplatin that relates to detects liquid-phase chip and detection method.

Background technology

The platinum class is a broad-spectrum anti-tumor medicine commonly used at present, has become indispensable medicine in the cancer chemotherapy.Developed at present and be the s-generation of representative and be the third generation platinum medicine of representative with the oxaliplatin with the carboplatin.The antitumor action of platinum medicine is by acting on DNA, forms the Pt-DNA binding substances, causes in the chain of DNA and interchain linkage, thereby suppresses the synthetic of DNA and duplicate.Though the curative effect of platinum medicine extensively approved, the individual difference of drug effect is very big and in various degree toxic side effect arranged after patient's medication.Its side effect mainly is a Digestive tract toxicity, and common also can have renal toxicity, liver toxicity and bone marrow depression for feeling sick, vomitting, and ototoxicity and neurotoxicity are lighter.

Vast amount of clinical is verified, and the expression level of the curative effect/toxic side effect of platinum medicine and patient's body inner nucleotide excision reparation cross complementary group 1 (ERCC1), hereditary breast cancer and ovarian cancer tumor susceptibility gene BRCA1 is in close relations.

(1) platinum medicine and nucleotide excision are repaired cross complementary group 1 (ERCC1)

ERCC1 is important member in the circumscribed reparation of the nucleic acid family, participates in the cutting and the damage identification of DNA chain.Disappearance has the tumour cell of function ERCC1 can not repair dna damage that cis-platinum causes and death; Otherwise ERCC1 mRNA high expression level then causes the resistance of platinum medicine.Studies show that in the middle and advanced stage nonsmall-cell lung cancer of excision ERCC1 low express accept gemcitabine and cisplatin combined chemotherapy after lifetime obviously prolong, ERCC1 expresses negative patients with lung cancer to accept adjuvant chemotherapy significantly benefited simultaneously.In addition, ERCC1 mRNA hangs down patient's median survival interval of expression apparently higher than the high expression level person in the progressive stage nonsmall-cell lung cancer.Research has confirmed that ERCC1 participates in nonsmall-cell lung cancer platinum-based chemotherapy medicine resistance and takes place, with the lung cancer curative effect be negative correlation lifetime; The low expresser of ERCC1 mRNA shows good yield to the adjuvant chemotherapy that contains the platinum-based chemotherapy medicine, thereby the expression level of ERCC1mRNA can be used as one of drug-fast key index of platinum medicine.

(2) platinum medicine, anti-microtubule class medicine and hereditary breast cancer and ovarian cancer tumor susceptibility gene BRCA1

Platinum compound is the tumor chemotherapeutic drug of the most normal use, because platinum medicine suppresses cell fission by broad incorporation DNA and realize anticancer purpose, so the use of platinum medicine damages proper splitting cell in patient's body inevitably, causes toxic side effect.A large amount of clinical studyes are verified: the BRCA1 mrna expression is closely related in the curative effect of platinum medicine and the tumor tissues, and promptly the patient that the BRCA1 gene expression dose is low is to the platinum medicine sensitivity, otherwise the high patient of expression level shows resistance.

Anti-microtubule based chemotherapy medicine is to act on the cell microtubule, forms by influencing spindle body, thus a based chemotherapy medicine of inhibition cell mitogen.The anticancer spectrum of anti-microtubule class medicine is very wide, is to use antitumor drug the most widely.But in clinical application, but occur the efficient low and big problem of side effect of treatment often, and have significant individual difference.Vast amount of clinical shows that the mRNA expression level of BRCA1 gene is closely related in the curative effect of anti-microtubule medicine and the tumor tissues, and promptly the patient that the BRCA1 gene expression dose is high resists microtubule class medicaments insensitive, otherwise the low patient of expression level shows resistance.

Show that in clinical study the mRNA expression level of BRCA1 is relevant with the curative effect of platinum class and paclitaxel treatment to ovarian cancer patients.The mRNA of BRCA1 is low to express and the patient of medium expression accepts lifetime behind the platinum-based chemotherapy than high expression level patient phenomenal growth, and in the patient of BRCA1 mRNA high expression level, the patient that the ratio of accepting taxol/platinum-based chemotherapy is only accepted platinum-based chemotherapy prolongs lifetime.

(3) housekeeping gene B2M (B2M), TfR (TFRC) and TATA frame conjugated protein (TBP)

But housekeeping gene is stably express in vivo generally, but because this stably express is relative, under certain pathological state or medicining condition, the expression of the housekeeping gene that some is commonly used also can change.The gene that is chosen in stably express in our institute's research object is made housekeeping gene, and is most important to the accuracy that guarantees detected result.Therefore, the present invention screens from the housekeeping gene commonly used of some amount, determines 3 suitable housekeeping genes, is respectively: B2M (B2M), TfR (TFRC), TATA frame conjugated protein (TBP).

Dependency based on above expression of gene level and therapeutic effectiveness of platinum medicaments, the expert recommends the patient before accepting the platinum medicine treatment, the detection of the mrna expression that should be correlated with, help the clinician to formulate the personalized medicine scheme according to patient's individual difference, to improve curative effect of medication, reduce the generation of poisonous side effect of medicine.

At present, the technology that the mRNA expression level of gene is detected mainly contains: reverse transcription quantitative PCR method, real-time fluorescence quantitative PCR method, RNA in-situ nucleic acid hybridization (RISH) etc.Wherein, RISH is mainly used in the analysis tissue or intracellular rna distributes with the expression of understanding specific gene, and its process is long, complex operation, is not suitable for clinical application; And reverse transcription quantitative PCR method and real-time fluorescence quantitative PCR method, at first, these two kinds of methods all need to carry out RNA extracting, reverse transcription, and it is big that the result of detection is influenced by the RNA degraded; Secondly, these two kinds of methods are only carried out pcr amplification by a pair of primer, are easy to generate non-specific binding, thereby cause the false positive height; Once more, these two kinds of methods generally have only a housekeeping gene to compare, and its accuracy is subjected to the influence of pathological state easily; Therefore, reverse transcription quantitative PCR method and real-time fluorescence quantitative PCR method all exist the technological deficiency that is difficult to overcome as clinical application.

Liquid-phase chip technology is utilized the carrier of polystyrene microsphere as reaction, in the manufacturing processed of microballoon, mixes the staining agent of different ratios, thereby forms the microballoon of 100 kinds of different colours codings.Thereby different microballoon covalent attachment realize the nearly detection of running simultaneously of material to be checked in 100 at the probe molecule of difference thing to be detected.Therefore, we adopt liquid-phase chip technology can detect the mRNA expression level of a plurality of genes simultaneously, have realized the high-throughput that detects, and have improved detection efficiency greatly.

Technical scheme

One of purpose of the present invention provides the mrna expression Liquid Detection chip relevant with therapeutic effectiveness of platinum medicaments, and this liquid-phase chip can be used for detecting the expression level of following 5 kinds of target genes: ERCC1, BRCA1, B2M, TFRC, TBP.

The technical scheme that realizes above-mentioned purpose is as follows:

A kind of mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip mainly includes:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end can with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.5, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.6-SEQ.NO.10 more than 2 or 2 and/or at the BRCA1 gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2; And at the B2M gene be selected among the SEQ.NO.17-SEQ.NO.21 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.22-SEQ.NO.26 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.27-SEQ.NO.31 more than 2 or 2;

(3) amplification extension probes, every amplification extension probes comprise 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5,3 ' end also is modified with vitamin H; The specific sequence P4 of described amplification extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.32-SEQ.NO.41 more than 5 or 5 and/or at the BRCA1 gene be selected among the SEQ.NO.42-SEQ.NO.53 more than 5 or 5; And at the B2M gene be selected among the SEQ.NO.54-SEQ.NO.63 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.64-SEQ.NO.73 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.74-SEQ.NO.83 more than 5 or 5; The described P5 of described P5 is for existing hairpin structure, do not form dimer between probe interior and probe, do not have mispairing, and P1, P2, P3, P4 and total mRNA between all do not have the sequence of non-specific binding.

Perhaps, mainly include:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end can with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.5, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.6-SEQ.NO.10 more than 2 or 2 and/or at the BRCA1 gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2; And at the B2M gene be selected among the SEQ.NO.17-SEQ.NO.21 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.22-SEQ.NO.26 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.27-SEQ.NO.31 more than 2 or 2;

(3) amplification extension probes, every the amplification extension probes comprise 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5; The specific sequence P4 of described amplification extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.32-SEQ.NO.41 more than 5 or 5 and/or at the BRCA1 gene be selected among the SEQ.NO.42-SEQ.NO.53 more than 5 or 5; And at the B2M gene be selected among the SEQ.NO.54-SEQ.NO.63 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.64-SEQ.NO.73 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.74-SEQ.NO.83 more than 5 or 5; Described P5 is for existing hairpin structure, do not form dimer between probe interior and probe, do not have mispairing, and P1, P2, P3, P4 and total mRNA between all do not have the sequence of non-specific binding; With

(4) label probe, described label probe have the sequence with amplification extension probes P5 complementary pairing, and end has biotin modification.

Preferably, also include padding sequence, comprising: at the SEQ.NO.84 of ERCC1 gene; One or more among the SEQ.NO.85-SEQ.NO.86 that be selected from the BRCA1 gene; One or more among the SEQ.NO87-SEQ.NO.94 that be selected from the B2M gene; And/or at one or more among the SEQ.NO.95-SEQ.NO.98 that be selected from of TFRC gene.

Preferably, described spacerarm sequence is 5-10 T; Described P5 consists of CCTATGCCTCCCGTGTCTA.

Another object of the present invention provides a kind of method that detects the mRNA expression of gene associated with treatment effect of carboplatin level.

Concrete scheme is as follows:

A kind of method that detects the mRNA expression of gene associated with treatment effect of carboplatin level is used each described liquid-phase chip of claim 1-4, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine complementary combination with the P1 that supports probe, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The amplification extension probes has biotin labeling, amplifies thereby the P4 of amplification extension probes and target mRNA specific combination realize the cascade of target mRNA signal;

(3) product and the Streptavidin-phycoerythrin of step (two) react;

(4) detect by fluorescence detector.

Perhaps, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine complementary combination with the P1 that supports probe, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The P4 and the target mRNA specific combination of amplification extension probes;

(3) add label probe again, label probe has biotin labeling, and 50 ℃ ± 1 ℃, the 600rpm concussion was hatched 1 hour, and label probe combines with the sequence P5 complementary pairing of amplification extension probes, thereby the cascade that realizes target mRNA signal is amplified;

(4) product and the Streptavidin-phycoerythrin of step (three) react;

(5) detect by fluorescence detector.

Major advantage of the present invention is:

(1) the designed various probes of the present invention can carry out hybridization under the reaction conditions of homogeneous, and do not have non-specific binding between the various probe substantially; Designed probe specificity in detection is good, signal to noise ratio is high.Simultaneously, being used in combination of multiple probe makes liquid-phase chip and detection method form an intact system of detection effect.Use liquid-phase chip of the present invention and detection method, the mRNA expression of gene associated with treatment effect of carboplatin level is detected, need not steps such as RNA extraction, reverse transcription, PCR, detected result is subjected in the sample quality influence of RNA less, has guaranteed the accuracy of detected result; On the other hand, compare (N 〉=3) with a plurality of housekeeping genes, detected result is not subject to the influence of pathological state, the accuracy height;

(2) the present invention uses is that the multidigit of probe is put the amplification that mode that special pairing, cascade amplify realizes signal, rather than the method for pcr amplification, improve detection signal, realized the specificity that detects, avoided the false positive of reverse transcription PCR and real-time fluorescence quantitative PCR technology.

(3) liquid-phase chip of the present invention and detection method are applicable to flesh tissue, all are suitable at paraffin embedding fixing organization, tissue slice.To mRNA detection method, the characteristics that operation is simple and easy, accuracy is high, specificity is high are arranged compared with other.

Embodiment

Known to the those skilled in the art, described spacerarm sequence is spaced apart for being used for support probe and microsphere surface, supporting extension probes and amplification extension probes inside also to have the spacerarm sequence, by between probe sequence and amido, probe interior is provided with the spacerarm sequence of suitable length, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n 〉=3) are as (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm (the preferred 2-4 of spacerarm length).Spacerarm of the present invention is preferably 5-10 T, in China, and the synthetic technology comparative maturity of T, cost is relatively low.

Embodiment 1 therapeutic effectiveness of platinum medicaments related objective mrna expression detects liquid phase chip reagent box, mainly includes:

One, coupling has the microballoon of support probe (SP)

Support probe to form by three parts, 5 ' end is and microballoon bonded amido end, 3 ' end is and supports extension probes bonded specific sequence P1, the centre is the spacerarm sequence of 8 oligonucleotide T, each target gene is selected a kind of microballoon for use, and select a support probe for use, every kind of microballoon has different fluorescence-encoded.The support probe of each target gene is as shown in table 1:

The target gene that table 1 is different and select for use microballoon numbering, support probe (SP)

Gene	Microballoon number	SP sequence (5 ' → 3 ') NH 2+poly(dT)+P1	??SEQ?ID
				??ERCC1	??20	??5’NH 2-TTTTTTTT-ATCATCAATCACTTTA	??1
??BRCA1	??56	??5’NH 2-TTTTTTTT-CTTTTACATTCATTAC	??2
				??B2M	??22	??5’NH 2-TTTTTTTT-CTTTCTTTAATCTCAA	??3
??TFRC	??61	??5’NH 2-TTTTTTTT-CATTCAAATCTCAACT	??4
				??TBP	??33	??5’NH 2-TTTTTTTT-CAATTACTTCAAATCT	??5

All probes are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Synthetic is supported probe sterilization ddH ₂O is made into the stock solution of 100nmol/ml.The process of microballoon bag quilt is as follows:

Get 5 * 10 respectively ⁶The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic capture probe (100nmol/ml).EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.Support the microballoon of probe to be resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0), 1mmol/LEDTA] of 100ul being coated with after the washing, 2-8 ℃ keeps in Dark Place.

Two, with the support extension probes (SE) of target gene mRNA specific combination, amplification extension probes (AE)

1) supports that extension probes (SE) is to connect the sequence of supporting between probe (SP) and the target gene mRNA, SE is made up of three parts, 5 ' end be can with target gene mRNA bonded specific sequence P2, length is between 17nt to 27nt, 3 ' end is to pass through base complementrity bonded specific sequence P3 with supporting probe 5 ' end, and the centre is the spacerarm sequence of 5 oligonucleotide T.Each target gene designs 5 to 6 respectively and supports extension probes, to improve the specificity that detects.During use, at every kind of target gene, select to support more than 2 or 2 extension probes can finish detection, specificity and stability all fine (experimental data omission) are preferable over and use 5 to 6 to support extension probes, so that specificity reaches best.Every probe after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.The support extension probes (SE) of target gene sees Table 2.

The support extension probes (SE) of table 2 target gene

2) amplification extension probes (AE) is the sequence between linking objective gene mRNA and the signal detection component, AE is made up of three parts, 5 ' end be can with target gene mRNA bonded specific sequence P4,3 ' end be can with the sequence P5 of label probe complementary pairing, (when not using label probe to detect, then 3 ' end also is modified with vitamin H), the centre is the spacerarm sequence of 5 oligonucleotide T.Each target gene designs 10 to 12 amplification extension probes respectively, amplifies thereby detection signal is carried out cascade.Every probe after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.The amplification extension probes (AE) of target gene sees Table 3.The design of described P5 is according to the common practise of the probe design of this area, it is not for existing inner hairpin structure, do not form dimer between probe interior and probe, do not have mispairing, and all do not have non-specific binding between P1, P2, P3, P4 and the total mRNA, the preferred based composition of the present invention is CCTATGCCTCCCGTGTCTA.

During use, at every kind of target gene, the extension probes of selecting to increase more than 5 or 5 can be finished detection, and specificity and stability all fine (experimental data omission) are preferable over and use 10 or 12 amplification extension probes, so that specificity reaches best.

The amplification extension probes (AE) of table 3 target gene

2) padding sequence (FS) will not seal with the zone of SE, AE specific combination among the target gene mRNA, with the non-specific binding of vitamin H or label probe in the minimizing subsequent reactions, thereby reduce detection background.The Tm value of padding sequence should be approaching with SE, AE, as shown in table 4.

The padding sequence of table 4 target gene (FS)

Three, label probe (LP)

The probe of mark (LP) is made up of two portions, its 3 ' end be can with the complementary bonded sequence of amplification extension probes sequence P5,5 ' end has biotin labeling, amplifies by combining the cascade that realizes target mRNA signal with the amplification extension probes.

It is as follows to the prescription of the described various solution of detection of clinical sample that embodiment 2 utilization mRNA expression of gene associated with treatment effect of carboplatin detect liquid-phase chip:

One, lysed sample discharges total RNA

1. FFPE tumor tissue section is scraped from slide glass, put into the 1.5ml centrifuge tube, add 300ul homogenate buffer and 3ul Proteinase K (50ug/ul), mix, 65 ℃ of digestion 2h.

2. the sample of digestion behind the 2h, under the room temperature 13, centrifugal 5 minutes of 000rpm, clear soln is transferred in the new 1.5ml centrifuge tube standby in the middle of drawing.(still do not clarify as solution, then repeat this step 1～2 time.)

Two, target mRNA is fixed on the microballoon by combining with probe specificity.

1. take out the probe working fluid and melt,, place on ice at once then 95 ℃ of sex change 5 minutes in room temperature.

2. according to the form below preparation working fluid mixes

3. the above-mentioned working fluid branch for preparing is filled on the hybridization plate 60ul/ hole.Then the above-mentioned sample of handling well is added respectively in the hybridization plate with the 40ul/ hole; Blank adds homogenate buffer 40ul/ hole.Sealing hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation (16-22h).

4. use washing buffer liquid wetting filter plate 1 minute, remove lavation buffer solution.

5. will hybridize plate and take out centrifugal 1 minute of 500 * g.Reaction solution all is transferred in the filter plate.

6. remove solution, with 200ul lavation buffer solution washing 3 times.

Three, by the multistep hybridization target RNA signal is amplified

1. add label probe working fluid 100ul/ hole.50 ℃ ± 1 ℃, the 600rpm concussion was hatched 1 hour.

2. remove solution, with 200ul washings washing 2 times.

Four, combine with SA-PE, and on the liquid-phase chip reading apparatus, detect

1. add SA-PE working fluid 100ul/ hole, 600rpm concussion incubated at room 30min.

2. remove solution,, blot the filter plate bottom with thieving paper with 200ul SA-PE washings washing 2 times.

3. add SA-PE washings 130ul/ hole.Room temperature, the 600rpm concussion was hatched 2 to 5 minutes.

4. reading of data on the liquid-phase chip instrument.

Five, data analysis and cutoff value defines

In 5 target genes, 2 of target genes are respectively: ERCC1, BRCA1;

3 of housekeeping genes are respectively: B2M, TFRC, TBP;

The fluorescent value that reads is carried out homogenization in accordance with the following methods to be handled:

Step 1: obtain raw data (MFI value)

Step 2: the MFI of sample deducts the MFI=net MFI of blank well N

Step 3: the net MFI value of three housekeeping genes of each sample is got geometrical mean respectively, obtains corresponding G1, G2, G3......Gn

Step 4: the net MFI of each sample target gene mRNA obtains corresponding N n divided by the Gn of correspondence.Nn is and gets rid of applied sample amount difference, can carry out relative expression quantity numerical value relatively at sample room.

In the present embodiment 10 routine samples are detected, detected result is as follows:

The relative expression quantity of the routine sample raw data of table 610 (MFI value) and ERCC1, BRCA1

Embodiment 3: the liquid-phase chip of different interval arm is to the detection of mRNA expression of gene associated with treatment effect of carboplatin level

One, the design (selection of spacerarm) of liquid-phase chip preparation

The support probe that detects liquid-phase chip with ERCC1 is an example, selects different spacerarms respectively for use, and specific design is as shown in table 7.Probe SP, SE and AE synthetic, SP sequence bag is described like embodiment 1 and embodiment 2 by microballoon, detection method.

Table 7 spacerarm and length thereof

The spacerarm kind	Length	Experimental group
			??poly(dT)	??8	??1
??poly(dA)	??8	??2
			??(CH2)n	??15	??3
??poly(TTG)	??3	??4

Two, sample detection

Adopt the liquid-phase chip of above-mentioned design preparation, sample 11-15 detected, detected result following (data are for detecting fluorescent value in the table) by embodiment 2 described testing processes and method:

Table 8 sample detection result (detect fluorescent value＜MFI value 〉)

??Sample	Experimental group 1	Experimental group 2	Experimental group 3	Experimental group 4
					??N	?8	?12	?18	?9
??11	?8596	?8645	?8589	?8649
					??12	?6859	?6845	?6829	?6798
??13	?6591	?6488	?6558	?6524
					??14	?3652	?3695	?3625	?3678
??15	?5625	?5698	?5627	?5675

The detection fluorescent value of 4 groups of designs is carried out statistical analysis, prove that the detection effect of 4 groups of designs does not have difference.Therefore, the design of these 4 kinds of spacerarms is equivalent.

Other is at the liquid-phase chip of supporting extension probes, intervening sequence that the amplification extension probes is inner different, and its result is still reliable and stable, and concrete data are omitted.

Embodiment 4: unlike signal detected components liquid-phase chip is to the detection of mRNA expression of gene associated with treatment effect of carboplatin level

One, the design (signal detection component) of liquid-phase chip preparation

The signal detection component has two kinds of selections, 1) 3 ' end of amplification extension probes 3 ' terminal sequence P5 has biotin labeling; 2) amplification extension probes 3 ' terminal sequence P5 combines by the base complementrity pairing with label probe (LP), and 5 ' of label probe end has biotin labeling simultaneously.These two kinds of signal detection components can realize that all signal amplifies, and detects normal signal.Wherein, the vitamin H activity of applying marking probe is more stable, and effect is more excellent.

Two kinds of signal detection components with the detection ERCC1 gene of described liquid-phase chip are example, and specific design is as shown in table 9.Be consisting of of described liquid-phase chip:

Experimental group 1: support probe link coupled microballoon, support extension probes, padding sequence with embodiment 1, the amplification extension probes has biotin labeling; There is not label probe;

Experimental group 2: support probe link coupled microballoon, support extension probes, padding sequence, label probe with embodiment 1, the amplification extension probes does not have biotin labeling.

Table 9 signal detection component

Two, sample detection

Adopt the liquid-phase chip of above-mentioned design preparation, detection method: experimental group 2 detects sample 16-20 with embodiment 2 described testing processes and method, experimental group 1 described detection method is omitted (three, target RNA signal is amplified by hybridization) this step, and all the other are with embodiment 2.

Table 10 sample detection result (detect fluorescent value＜MFI value 〉)

??Sample	Experimental group 1	Experimental group 2
			??N	??9	??15
??16	??5968	??5989
			??17	??4658	??4689
??18	??8569	??8596
			??19	??3596	??3625
??20	??5681	??5698

The detection fluorescent value of two groups of designs is carried out statistical analysis, prove that the detected result of two groups of designs does not have difference, therefore, these two kinds of signal detection components are equivalent to the detection of signal.Wherein, the signal detection component of applying marking probe, because the activity of its vitamin H is more stable, effect is more excellent.

More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.

Sequence table

＜110〉Guangzhou Yishan Biotechnology Co., Ltd.

＜120〉gene mRNA expression of therapeutic effectiveness of platinum medicaments prediction detects liquid-phase chip and detection method

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ggcgaggatc?aatgtgcagt?c??????????????????????21

<210>9

<211>19

<212>DNA

＜213〉artificial sequence

<400>9

gaggtccgct?ggtttctgc?????????????????????????19

<210>10

<211>23

<212>DNA

＜213〉artificial sequence

<400>10

caggagggtc?tgactgtccg?ttt????????????????????23

<210>11

<211>21

<212>DNA

＜213〉artificial sequence

<400>11

gagtgagagg?agctcccagg?g??????????????????????21

<210>12

<211>25

<212>DNA

＜213〉artificial sequence

<400>12

caagcagaaa?atctttaagg?gaccc??????????????????25

<210>13

<211>26

<212>DNA

＜213〉artificial sequence

<400>13

gcctgaatag?aaagaatagg?gctgat?????????????????26

<210>14

<211>25

<212>DNA

＜213〉artificial sequence

<400>14

tgcctttgtg?aacacagggt?tttag??????????????????25

<210>15

<211>25

<212>DNA

＜213〉artificial sequence

<400>15

actgcccaag?gactattctg?acttt??????????????????25

<210>16

<211>23

<212>DNA

＜213〉artificial sequence

<400>16

agtgctggga?ttacaggcgt?gag????????????????????23

<210>17

<211>20

<212>DNA

＜213〉artificial sequence

<400>17

aaggccacgg?agcgagacat????????????????????????20

<210>18

<211>22

<212>DNA

＜213〉artificial sequence

<400>18

ccattctctg?ctggatgacg?tg?????????????????????22

<210>19

<211>24

<212>DNA

＜213〉artificial sequence

<400>19

gctgaaagac?aagtctgaat?gctc???????????????????24

<210>20

<211>25

<212>DNA

＜213〉artificial sequence

<400>20

ttaactatct?tgggctgtga?caaag??????????????????25

<210>21

<211>25

<212>DNA

＜213〉artificial sequence

<400>21

ggagacagca?ctcaaagtag?aatta??????????????????25

<210>22

<211>24

<212>DNA

＜213〉artificial sequence

<400>22

acaatagccc?aagtagccaa?tcat???????????????????24

<210>23

<211>22

<212>DNA

＜213〉artificial sequence

<400>23

tcggttcctg?ccagtctctc?ac?????????????????????22

<210>24

<211>24

<212>DNA

＜213〉artificial sequence

<400>24

tctccgacaa?ctttctcttc?aggt???????????????????24

<210>25

<211>21

<212>DNA

＜213〉artificial sequence

<400>25

ccagcctcac?gagggacata?t??????????????????????21

<210>26

<211>23

<212>DNA

＜213〉artificial sequence

<400>26

acgccagact?ttgctgagtt?taa????????????????????23

<210>27

<211>24

<212>DNA

＜213〉artificial sequence

<400>27

cgaagtgcaa?tggtctttag?gtca???????????????????24

<210>28

<211>23

<212>DNA

＜213〉artificial sequence

<400>28

cgtggttcgt?ggctctctta?tcc????????????????????23

<210>29

<211>22

<212>DNA

＜213〉artificial sequence

<400>29

tattttcttg?ctgccagtct?gg?????????????????????22

<210>30

<211>24

<212>DNA

＜213〉artificial sequence

<400>30

catcacagct?ccccaccata?ttct???????????????????24

<210>31

<211>26

<212>DNA

＜213〉artificial sequence

<400>31

caattctggg?tttgatcatt?ctgtag?????????????????26

<210>32

<211>21

<212>DNA

＜213〉artificial sequence

<400>32

gcccagcaca?tagtcgggaa?t??????????????????????21

<210>33

<211>20

<212>DNA

＜213〉artificial sequence

<400>33

gggtgcaggt?tgtggtagcg????????????????????????20

<210>34

<211>19

<212>DNA

＜213〉artificial sequence

<400>34

cagccgccca?tggatgtag?????????????????????????19

<210>35

<211>21

<212>DNA

＜213〉artificial sequence

<400>35

aaggcgaagt?tcttccccag?g??????????????????????21

<210>36

<211>24

<212>DNA

＜213〉artificial sequence

<400>36

cacatcttag?ccagctcctt?gagg???????????????????24

<210>37

<211>23

<212>DNA

＜213〉artificial sequence

<400>37

gccttgtagg?tctccaggta?ccg????????????????????23

<210>38

<211>25

<212>DNA

＜213〉artificial sequence

<400>38

gacgaagtcc?tgctctagct?tctcc??????????????????25

<210>39

<211>25

<212>DNA

＜213〉artificial sequence

<400>39

gacttcacgg?tggtcagaca?ttcag??????????????????25

<210>40

<211>25

<212>DNA

＜213〉artificial sequence

<400>40

tgagctgttc?cagagatcca?aatgt??????????????????25

<210>41

<211>23

<212>DNA

＜213〉artificial sequence

<400>41

gccagatctt?ctcttgatgc?ggc????????????????????23

<210>42

<211>26

<212>DNA

＜213〉artificial sequence

<400>42

tttagtagcc?aggacagtag?aaggac?????????????????26

<210>43

<211>25

<212>DNA

＜213〉artificial sequence

<400>43

cagaagtcct?tttcaggctg?atgta??????????????????25

<210>44

<211>22

<212>DNA

＜213〉artificial sequence

<400>44

atggcagatt?tccaagggag?ac?????????????????????22

<210>45

<211>23

<212>DNA

＜213〉artificial sequence

<400>45

caattggtgg?cgtttaaatg?gtt????????????????????23

<210>46

<211>21

<212>DNA

＜213〉artificial sequence

<400>46

cttccagccc?taagccaaca?a??????????????????????21

<210>47

<211>23

<212>DNA

＜213〉artificial sequence

<400>47

aaccagctat?tctcttgagg?cca????????????????????23

<210>48

<211>24

<212>DNA

＜213〉artificial sequence

<400>48

ttccattgaa?gggtctgact?ctct???????????????????24

<210>49

<211>24

<212>DNA

＜213〉artificial sequence

<400>49

tcacataatc?gatcccaagc?actc???????????????????24

<210>50

<211>23

<212>DNA

＜213〉artificial sequence

<400>50

cccaatgttc?cactccaaca?ttt????????????????????23

<210>51

<211>23

<212>DNA

＜213〉artificial sequence

<400>51

accatgccca?ggtttcaagt?ttc????????????????????23

<210>52

<211>21

<212>DNA

＜213〉artificial sequence

<400>52

tccagtgatc?tgcccacctt?g??????????????????????21

<210>53

<211>21

<212>DNA

＜213〉artificial sequence

<400>53

gtttcaccat?gttggccagg?c??????????????????????21

<210>54

<211>17

<212>DNA

＜213〉artificial sequence

<400>54

ttcaggaatg?cccgcca???????????????????????????17

<210>55

<211>23

<212>DNA

＜213〉artificial sequence

<400>55

ccaggccaga?aagagagagt?agc????????????????????23

<210>56

<211>23

<212>DNA

＜213〉artificial sequence

<400>56

cctgaatctt?tggagtacgc?tgg????????????????????23

<210>57

<211>24

<212>DNA

＜213〉artificial sequence

<400>57

ggatgaaacc?cagacacata?gcaa???????????????????24

<210>58

<211>24

<212>DNA

＜213〉artificial sequence

<400>58

agtgggggtg?aattcagtgt?agta???????????????????24

<210>59

<211>23

<212>DNA

＜213〉artificial sequence

<400>59

tcacacggca?ggcatactca?tct????????????????????23

<210>60

<211>22

<212>DNA

＜213〉artificial sequence

<400>60

gctgcttaca?tgtctcgatc?cc?????????????????????22

<210>61

<211>20

<212>DNA

＜213〉artificial sequence

<400>61

tgcggcatct?tcaaacctcc????????????????????????20

<210>62

<211>25

<212>DNA

＜213〉artificial sequence

<400>62

gcaacctgct?cagatacatc?aaaca??????????????????25

<210>63

<211>23

<212>DNA

＜213〉artificial sequence

<400>63

cctctaagtt?gccagccctc?cta????????????????????23

<210>64

<211>25

<212>DNA

＜213〉artificial sequence

<400>64

atagtcccat?agcagatact?tccac??????????????????25

<210>65

<211>25

<212>DNA

＜213〉artificial sequence

<400>65

caatcaagaa?aaagacgatc?acagc??????????????????25

<210>66

<211>23

<212>DNA

＜213〉artificial sequence

<400>66

ctcagttttt?ggttctaccc?ctt????????????????????23

<210>67

<211>21

<212>DNA

＜213〉artificial sequence

<400>67

ctcctggctc?ctccctcact?g??????????????????????21

<210>68

<211>19

<212>DNA

＜213〉artificial sequence

<400>68

cgacgtgctg?cagggaagt?????????????????????????19

<210>69

<211>22

<212>DNA

＜213〉artificial sequence

<400>69

gctggtgaag?tctgtgctgt?cc?????????????????????22

<210>70

<211>22

<212>DNA

＜213〉artificial sequence

<400>70

ttttcattca?gcagcttgat?gg?????????????????????22

<210>71

<211>22

<212>DNA

＜213〉artificial sequence

<400>71

cgagttttga?gcgctgtctt?tg?????????????????????22

<210>72

<211>24

<212>DNA

＜213〉artificial sequence

<400>72

caggattctc?caccaggtaa?acaa???????????????????24

<210>73

<211>23

<212>DNA

＜213〉artificial sequence

<400>73

gttgcagcct?tactatacgc?cac????????????????????23

<210>74

<211>19

<212>DNA

＜213〉artificial sequence

<400>74

gcagctgcgg?tacaatccc?????????????????????????19

<210>75

<211>25

<212>DNA

＜213〉artificial sequence

<400>75

tacaaccaag?attcactgtg?gatac??????????????????25

<210>76

<211>22

<212>DNA

＜213〉artificial sequence

<400>76

gattatattc?ggcgtttcgg?gc?????????????????????22

<210>77

<211>21

<212>DNA

＜213〉artificial sequence

<400>77

atgattaccg?cagcaaaccg?c??????????????????????21

<210>78

<211>22

<212>DNA

＜213〉artificial sequence

<400>78

gcacaccatt?ttcccagaac?tg?????????????????????22

<210>79

<211>23

<212>DNA

＜213〉artificial sequence

<400>79

ctgttcttca?ctcttggctc?ctg????????????????????23

<210>80

<211>24

<212>DNA

＜213〉artificial sequence

<400>80

acccaacttc?tgtacaactc?tagc???????????????????24

<210>81

<211>25

<212>DNA

＜213〉artificial sequence

<400>81

tcttgaagtc?caagaactta?gctgg??????????????????25

<210>82

<211>22

<212>DNA

＜213〉artificial sequence

<400>82

gggtgagcac?aaggccttct?aa?????????????????????22

<210>83

<211>26

<212>DNA

＜213〉artificial sequence

<400>83

caggaaataa?ctctggctca?taacta?????????????????26

<210>84

<211>17

<212>DNA

＜213〉artificial sequence

<400>84

cagcttcctc?ggggctc???????????????????????????17

<210>85

<211>26

<212>DNA

＜213〉artificial sequence

<400>85

tcttctcagg?tgaaaaatta?ccataa?????????????????26

<210>86

<211>26

<212>DNA

＜213〉artificial sequence

<400>86

tcatttctaa?tacctgcctc?agaatt?????????????????26

<210>87

<211>17

<212>DNA

＜213〉artificial sequence

<400>87

cggcccgaat?gctgtca???????????????????????????17

<210>88

<211>22

<212>DNA

＜213〉artificial sequence

<400>88

cagtaagtca?acttcaatgt?cg?????????????????????22

<210>89

<211>23

<212>DNA

＜213〉artificial sequence

<400>89

ctttttcaat?tctctctcca?ttc????????????????????23

<210>90

<211>20

<212>DNA

＜213〉artificial sequence

<400>90

agagatagaa?agaccagtcc????????????????????????20

<210>91

<211>23

<212>DNA

＜213〉artificial sequence

<400>91

agaatttgga?attcatccaa?tcc????????????????????23

<210>92

<211>23

<212>DNA

＜213〉artificial sequence

<400>92

catatcaata?ttaaaaagca?agc????????????????????23

<210>93

<211>23

<212>DNA

＜213〉artificial sequence

<400>93

ctacattttg?tgcataaagt?gta????????????????????23

<210>94

<211>23

<212>DNA

＜213〉artificial sequence

<400>94

gaagatcatg?tccatgttaa?cat????????????????????23

<210>95

<211>23

<212>DNA

＜213〉artificial sequence

<400>95

cgcaagattt?tcatcttttt?gag????????????????????23

<210>96

<211>23

<212>DNA

＜213〉artificial sequence

<400>96

cacgaaattg?attttcaaca?tac????????????????????23

<210>97

<211>22

<212>DNA

＜213〉artificial sequence

<400>97

ctgaatctta?acaaaatgtt?ga?????????????????????22

<210>98

<211>23

<212>DNA

＜213〉artificial sequence

<400>98

ctaccgttct?tatcaactat?gat????????????????????23

<210>99

<211>19

<212>DNA

＜213〉artificial sequence

<400>99

tagacacggg?aggcatagg?????????????????????????19

Claims (10)

1. a mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip is characterized in that, mainly includes:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end can with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.5, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.6-SEQ.NO.10 more than 2 or 2 and/or at the BRCA1 gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2; And at the B2M gene be selected among the SEQ.NO.17-SEQ.NO.21 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.22-SEQ.NO.26 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.27-SEQ.NO.31 more than 2 or 2;

(3) amplification extension probes, every amplification extension probes comprise 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5,3 ' end also is modified with vitamin H; The specific sequence P4 of described amplification extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.32-SEQ.NO.41 more than 5 or 5 and/or at the BRCA1 gene be selected among the SEQ.NO.42-SEQ.NO.53 more than 5 or 5; And at the B2M gene be selected among the SEQ.NO.54-SEQ.NO.63 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.64-SEQ.NO.73 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.74-SEQ.NO.83 more than 5 or 5; Described P5 is for existing hairpin structure, do not form dimer between probe interior and probe, do not have mispairing, and P1, P2, P3, P4 and total mRNA between all do not have the sequence of non-specific binding.

2. mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip according to claim 1, it is characterized in that, also include padding sequence, comprising: at the SEQ.NO.84 of ERCC1 gene, at the BRCA1 gene be selected from one or more among the SEQ.NO.85-SEQ.NO.86, at the B2M gene be selected from one or more among the SEQ.NO87-SEQ.NO.94 and/or at one or more among the SEQ.NO.95-SEQ.NO.98 that be selected from of TFRC gene.

3. mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip according to claim 1 and 2 is characterized in that, described spacerarm sequence is 5-10 T.

4. mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip according to claim 1 is characterized in that, described P5 consists of CCTATGCCTCCCGTGTCTA.

5. a mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip is characterized in that, mainly includes:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end can with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.5, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.6-SEQ.NO.10 more than 2 or 2 and/or at the BRCA1 gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2; And at the B2M gene be selected among the SEQ.NO.17-SEQ.NO.21 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.22-SEQ.NO.26 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.27-SEQ.NO.31 more than 2 or 2;

(3) amplification extension probes, every the amplification extension probes comprise 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5; The specific sequence P4 of described amplification extension probes includes: at the ERCC1 gene be selected among the SEQ.NO.32-SEQ.NO.41 more than 5 or 5 and/or at the BRCA1 gene be selected among the SEQ.NO.42-SEQ.NO.53 more than 5 or 5; And at the B2M gene be selected among the SEQ.NO.54-SEQ.NO.63 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.64-SEQ.NO.73 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.74-SEQ.NO.83 more than 5 or 5; Described P5 is for existing hairpin structure, do not form dimer between probe interior and probe, do not have mispairing, and P1, P2, P3, P4 and total mRNA between all do not have the sequence of non-specific binding; With

(4) label probe, described label probe have the sequence with amplification extension probes P5 complementary pairing, and end has biotin modification.

6. mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip according to claim 5, it is characterized in that, also include padding sequence: comprise, at the SEQ.NO.84 of ERCC1 gene, at the BRCA1 gene be selected from one or more among the SEQ.NO.85-SEQ.NO.86, at the B2M gene be selected from one or more among the SEQ.NO.87-SEQ.NO.94 and/or at one or more among the SEQ.NO.95-SEQ.NO.98 that be selected from of TFRC gene.

7. according to claim 5 or 6 described mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chips, it is characterized in that described spacerarm sequence is 5-10 T.

8. mRNA expression of gene associated with treatment effect of carboplatin level detection liquid-phase chip according to claim 5 is characterized in that, described P5 consists of CCTATGCCTCCCGTGTCTA.

9. a method that detects the mRNA expression of gene associated with treatment effect of carboplatin level is characterized in that, uses each described liquid-phase chip of claim 1-4, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine complementary combination with the P1 that supports probe, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The amplification extension probes has biotin labeling, amplifies thereby the P4 of amplification extension probes and target mRNA specific combination realize the cascade of target mRNA signal;

(3) product and the Streptavidin-phycoerythrin of step (two) react;

(4) detect by fluorescence detector.

10. a method that detects the mRNA expression of gene associated with treatment effect of carboplatin level is characterized in that, uses each described liquid-phase chip of claim 5-8, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine complementary combination with the P1 that supports probe, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The P4 and the target mRNA specific combination of amplification extension probes;

(3) add label probe again, label probe has biotin labeling, and 50 ℃ ± 1 ℃, the 600rpm concussion was hatched 1 hour, and label probe combines with the sequence P5 complementary pairing of amplification extension probes, thereby the cascade that realizes target mRNA signal is amplified;

(4) product and the Streptavidin-phycoerythrin of step (three) react;

(5) detect by fluorescence detector.