CN103305600A - Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof - Google Patents
Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof Download PDFInfo
- Publication number
- CN103305600A CN103305600A CN2013100327682A CN201310032768A CN103305600A CN 103305600 A CN103305600 A CN 103305600A CN 2013100327682 A CN2013100327682 A CN 2013100327682A CN 201310032768 A CN201310032768 A CN 201310032768A CN 103305600 A CN103305600 A CN 103305600A
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr
- sample
- kinds
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for synchronously detecting related gene expression level of 14 antitumor drugs by using a paraffin embedding biopsy sample, and a detection method thereof. The kit comprises DEPC (diethylpyrocarbonate) water, 5*RT buffer solution, a reverse transcription primer, a reverse transcription enzyme, an X solution, 10*PCR buffer solution, a PCR primer, 25mM magnesium chloride solution, DNA (deoxyribonucleic acid) polymerase and a positive reference substance; the reverse transcription primer comprises related genes of the 14 antitumor drugs, and RT amplification primers of an RNA (ribonucleic acid) internal reference; gene sequences are shown in SEQ ID NO.1 to NO.18; the PCR primer includes related genes of the 14 antitumor drugs, and positive and negative PCR amplification primers of a DNA internal reference; the gene sequences are shown in SEQ ID NO.19 to NO.38. The kit has the advantages of strong specificity, high sensitivity, high flux, strong reliability, low cost and absence of false-negative result.
Description
Technical field
The present invention relates to test kit and the detection method thereof of antitumor medication related gene expression level, especially relate to a kind of test kit and detection method thereof of using 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection based on GeXP multiple gene expression genetic analysis systems.
Background technology
Recent years, pharmacogenetics/pharmacogenomics has obtained breakthrough in the research of the aspects such as antitumor drug mechanism of action, finds that some antitumor drug is to the lethal effect of tumour cell and specifically expression and/or the polymorphism significant correlation of a kind of (or one group) gene.By the detection of genes involved, the curative effect of prediction chemotherapy and targeted drug selects suitable medicine to carry out Individual Chemotherapy, the choose reasonable that become and improved curative effect, reduces futile treatment.
Individualized treatment is the genetics characteristics according to the patient, the method that adopts special and best pharmaceutical admixtures to treat.A large amount of clinical datas show, the target gene mrna expression level such as ERCC1/RRM1/TYMS/TUBB3 can predict respectively that the patient is to the reaction of platinum class/gemcitabine/fluorouracil/Common Chemotherapy medicines such as anti-microtubule class in the tumor tissues.According to the detected result of mrna expression level in the specimens, work out the individualized treatment scheme, help to select to be fit to patient's chemotherapeutics, improve the specific aim for the treatment of.Select chemotherapeutics can avoid invalid or harmful chemotherapy, valuable treatment time and medical expense of saving by target detection, improve patients ' life quality.
At present, the method for traditional gene expression detection (mRNA level) has following multiple:
(1) PCR detection method: that uses at present often has real-time fluorescence quantitative PCR, immuno-PCR, a reverse transcription PCR etc., all is that the specific target gene for pathogenic agent detects.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of the method is that susceptibility is high, but and accurate quantification; The method also has its shortcoming, and flux is low: the expression level that once can only detect a gene; Cost is relatively high: because once detecting a gene, when a sample needs to detect a plurality of genetic expression simultaneously, must detect one by one, cost increases relatively.
(2) molecular hybridization---Northern hybridization and RNA in situ hybridization: complementary nucleotide sequence is called hybridization by the process that the Walson-Crick base pairing forms stable heteroduplex molecular dna molecule.Crossover process is high degree of specificity, can carry out specific target sequence to known array according to employed probe and detect.Molecular hybridization has the specificity of very high sensitivity and height, thereby this technology is used in the qualitative and quantitative analysis of specific gene sequence in the making, genome of screening, the restriction enzyme mapping of clone gene and the aspects such as diagnosis of disease widely in biology field.But its complex operation step, and technical requirements is higher; This technology flux is low simultaneously, once can only detect a goal gene.
(3) gene chips: gene chip is to pass through micro-processing technology, with ten hundreds of, and even the dna fragmentation of 1,000,000 particular sequence (gene probe), arrange regularly and be fixed on the upholders such as silicon chip, slide, a two-dimentional dna probe array that consists of, utilize the biological sample of this class chip and mark to hybridize, can carry out fast qualitative and quantitative analysis to the gene expression profile bioinformation of sample.The advantage of gene chips is: 1) high-flux parallel detects: when a sample needed to detect a plurality of gene expression dose simultaneously, once experiment can draw whole results; 2) easy and simple to handle quick: whole detection only needed 4-8 hour substantially can go out the result.But also have following shortcoming: 1) cost is higher: chip of each sample needs, and cost Da Yu $1000/ sample is unfavorable for large-scale promotion; Synthetic and the fixing more complicated of probe is particularly made highdensity probe array, is main rate-limiting step; 2) can not accurate quantification, poor repeatability; 3) sensitivity is lower and need the nucleic acid amount larger, because the kind of chip is more, is difficult to formulate a unified quality control standard in addition.
(4) transcribe the group order-checking: transcribe group (transcriptome) and broadly refer under a certain physiological condition, the set of all transcription products comprises messenger RNA(mRNA), ribosome-RNA(rRNA), transfer RNA and non-coding RNA in the cell; Refer to narrowly the set of all mRNA.Transcribe group order-checking and generally be ripe mRNA and ncRNA that the rna plymerase ii that carries out affinity purification with poly thymus pyrimidine (oligo-dT) is transcribed generation and carry out high-flux sequence.Comprehensively obtain rapidly a certain species certain organs or be organized in nearly all transcript under a certain state, reflect their expression level.With respect to traditional chip hybridization platform, transcribing the group order-checking need not in advance for the known array designing probe, can the integral body activity of transcribing of any species be detected, more exact figure signal is provided, higher detection flux and sensing range widely are the powerful tool that the group complicacy is transcribed at present further investigation.Along with the continuous progress of sequencing technologies, sequencing throughput is increasing, but its cost is always high, and common patient can't bear, and the complicated operation of order-checking, and the cycle is longer, wouldn't be applicable to clinical treatment at present.
GenomeLab
TMGeXP multiple gene expression genetic analysis systems forms based on capillary electrophoresis separation technology and the research and development of highly sensitive laser Induced Fluorescence Technology of Beckman company maturation, the kapillary display and design in a branch of 8 roads takes full advantage of the alignment characteristics of 96 orifice plates, has reduced cost and the complicacy of using larger display to bring.Adopt multiple PCR method, by Beckman Coulter dye marker, in same EP pipe, analyze simultaneously the expression of a plurality of genes, can fast and effeciently detect the expression situation of gene, overcome the defective that above-mentioned fluorescence quantitative PCR method exists, had the following advantages:
1, high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 30-40 site, can do simultaneously 192 reactions (such as 192 patient's samples, 30 kinds of diarrhea viruses of each sample detection, 30 sites), went out the result in one day; For co-infected patients, present method can disposablely provide accurate report, avoids undetected.
2, accuracy is strong: GeXP adopts capillary electrophoresis that the PCR product is carried out separation detection, non-specific amplification product, primer dimer and specific amplification products can be separated, at utmost reduce false positive;
3, susceptibility is high, as a result good reproducibility: the GeXP system has overcome the deviation that the unequal amplification of normal PCR amplification method causes, and has improved a cover goal gene is carried out quantitative speed and susceptibility, adopts laser induced fluorescence(LIF)-PMT, has hypersensitivity;
4, method is easy, and use economical: GeXP provides from a complete set of experimental programs such as reagent, multiple PCR primer design, result and quantitative expression spectrum analysis; The testing cost Shao Yu $50 of each sample is beneficial to large-scale promotion;
5, accurate quantification, handiness are strong: but accurate quantification pathogen gene copy number can be adjusted the target gene of detection at any time according to demand.
6, easily be automated: with regard to sample preparation, Biomek series automated fluid processing instrument can mate fully with GeXP analyser and Ampligrid amplification instrument, and integrated bar code reader has guaranteed that sample is followed the trail of and report the test accurately.
At present, both at home and abroad also not about based on the test kit of 14 kinds of antitumor medication related gene expression levels of synchronous detection of GeXP multiple gene expression genetic analysis systems and the correlative study report of detection method thereof.
Summary of the invention
Technical problem to be solved by this invention provides a kind of high specificity, highly sensitive, flux is high, reliability is strong, cost is low, without test kit and the detection method thereof based on 14 kinds of antitumor medication related gene expression levels of use specimens paraffin embedding slices sample synchronous detection of GeXP multiple gene expression genetic analysis systems of false negative result.
The present invention solves the problems of the technologies described above the technical scheme that adopts: a kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection, comprise DEPC water, 5 * RT damping fluid, the reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, the PCR primer, the 25mM magnesium chloride solution, archaeal dna polymerase and positive reference substance, described reverse transcription primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds in the following table 1 and the RT amplimer of RNA confidential reference items, described PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds in the following table 1, forward and reverse pcr amplification primer of the pcr amplification primer of RNA confidential reference items and DNA confidential reference items, gene order is as shown in table 1 below:
Table 1
Described X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; The reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
Described positive reference substance is the RNA mixture that extracts from tumour cell, and described RNA mixture comprises described 14 kinds of antitumor medication related gene mRNAs.
A kind of method of utilizing the antitumor medication related gene expression of the detection level of the test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection specifically may further comprise the steps:
(1) collecting sample and extract nucleic acid
Gather the separation and Culture thing of specimens specimens paraffin embedding slices sample, from the separation and Culture thing, extract nucleic acid;
(2) carry out the RT reaction take patient's nucleic acid as template
Get the nucleic acid samples RNA5 μ L of 5-20ng/ul, DEPC water 8 μ L, 5 * RT damping fluid, 4 μ L, RT primer solution 2 μ L join on the 96 hole sample panel behind the RT enzyme 1 μ L mixing and carry out reverse transcription, reaction conditions: 48 ° C1 minute; 42 ° C60 minute; 95 ° C5 minute; 4 ° of C are until collect the RT product; Wherein each RT primer concentration is 500nM in the RT primer solution, and described RT primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds and the RT amplimer of RNA confidential reference items, and gene order is shown in SEQ ID NO.1～NO.18 in the sequence table;
(3) carry out the PCR reaction take reverse transcription product as template
Get RT product 8.6 μ L, 10 * PCR damping fluid, 2 μ L, the magnesium chloride 4 μ L of 25mM, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L join the reaction of the enterprising performing PCR of 96 hole sample panel, reaction conditions behind the X solution 2 μ L mixings: 95 ° C10 minute; 94 ° of C30 seconds, 55 ° of C30 seconds, 70 ° C1 minute, circulate 35 times; 70 ° C1 minute; 4 ° of C are until collect the PCR product; Wherein said X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; The reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark, each PCR primer concentration is 200nM in the PCR primer solution, the PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds, the pcr amplification primer of RNA confidential reference items and forward and reverse pcr amplification primer of DNA confidential reference items, and gene order is shown in SEQ ID NO.19～NO.38 in the sequence table;
(4) GeXP genetic analyzer electrocapillary phoresis sample separation
Get PCR product 0.1-1 μ L, the sample-loading buffer 38.75 μ L that the GeXP genetic analyzer is supporting, DNA Marker0.5 μ L, join after one in mineral oil mixes on the 96 hole parting liquid plates and carry out the electrocapillary phoresis sample separation, the collection of illustrative plates that the software of GeXP genetic analyzer is obtained contrasts with standard diagram, obtains the expression level of antitumor medication genes involved.
Compared with prior art, the invention has the advantages that: a kind of test kit and detection method thereof of using 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection of the present invention, because this test kit has been introduced for the relevant gene of 14 antitumor medications, 4 designed specificity amplification primers such as reference gene, expression level according to these genes instructs the nearly medicining condition of 24 kinds of antitumor drugs, can finish the detection of 192 patient's samples within one day, both saved production cost and testing cost, and improved again detection efficiency and shortened the time; The use of four reference genes can be used for monitoring the efficient of whole reactive system, RT-PCR reaction and the quality of assessment RNA template, avoids false negative; The use of reaction confidential reference items can be used for monitoring the efficient of whole reactive system, PCR reaction, avoids false negative, has better sensitivity and specificity so that detect, thereby has avoided the not high problem of other detection method specificitys.
In sum, the present invention is based on test kit and the detection method thereof of 14 kinds of antitumor medication related gene expression levels of use specimens paraffin embedding slices sample synchronous detection of GeXP multiple gene expression genetic analysis systems, can detect for 14 antitumor medication genes involveds simultaneously, detection sensitivity is high, specificity is good, reduce the false positive rate of conventional pcr amplification, can also effectively solve the easy pollution problem of conventional PCR; Has the Noncompetitive internal comparison system, reliability is strong, without false negative result, utilizes GeXP genetic analysis systems sensitivity, accurate quantitative analysis, quick, high-throughout technical superiority, provide a kind of superior cheaply molecular diagnosis method for clinical, help chemotherapeutics to use safely and effectively.
Description of drawings
Fig. 1 is the electrocapillary phoresis sample separation result standard collection of illustrative plates of GeXP genetic analyzer.
Embodiment
Embodiment is described in further detail the present invention below in conjunction with accompanying drawing.
A kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection of the present invention comprises following reagent in this test kit:
1) RT primer (reverse transcription primer RT primer Mix)
2) PCR primer (PCR Primer Mix)
3) 25mM magnesium chloride (25mM MgCl2)
4) reversed transcriptive enzyme (Reverse transcripatase)
5) archaeal dna polymerase (Taq DNA Polymerase)
6) X solution (Solution X)
7) 10 * PCR damping fluid (10 * PCR Buffer)
8) 5 * RT damping fluid (5 * RT buffer)
9) positive control (Positive Control)
11) without RNA enzyme/DNA enzyme ultrapure water (Dnase/Rnase Free ddH2O)
12) positive reference substance
Above-mentioned reverse transcription primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds in the following table 1 and the RT amplimer of RNA confidential reference items, described PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds in the following table 1, the pcr amplification primer of RNA confidential reference items and forward and reverse pcr amplification primer of DNA confidential reference items, and gene order is as shown in table 1:
The multiple gene test oligonucleotide sequence of the antitumor medication genes involved of table 1
Above-mentioned X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; The reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
Above-mentioned positive reference substance is the RNA mixture that extracts from tumour cell, comprises above-mentioned 14 kinds of antitumor medication related gene mRNAs.
Expression level according to above-mentioned 14 kinds of antitumor medication genes involveds instructs the nearly medicining condition of 24 kinds of antitumor drugs, and is specific as follows:
(1) BRCA1: expression level and patient are proportionate to the susceptibility of platinum class and anti-microtubule class medicine;
(2) DPYD: expression level and 5-FU(5-Fluracil) susceptibility is negative correlation;
(3) EGFR: expression level and Cetuximab resistance are proportionate;
(4) PTEN: down-regulated expression is the potential sign of mammary cancer differentiation and transfer;
(5) ERCC1: expression level and therapeutic effectiveness of platinum medicaments are negative correlation;
(6) STMN1: it is better that low expression level is accepted taxanes/cis-platinum (drug combination that refers to taxol and cis-platinum) result for the treatment of; The high expression level patient tumors shifts risk;
(7) TUBB3: low expression level accepts taxanes/cis-platinum or the vinca result for the treatment of is better; The high expression level patient tumors shifts risk;
(8) RRM1: expression level and gemcitabine resistance are proportionate;
(9) VEGFR: expression level and rhuMAb-VEGF curative effect are proportionate;
(10) HER2: expression level and Trastuzumab curative effect are proportionate;
(11) TYMP: expression level and capecitabine curative effect are proportionate; The low patient of expression level has preferably prognosis;
(12) TYMS: expression level and 5-FU curative effect are proportionate;
(13) PDGFR: expression level variation prediction Xarelto curative effect;
(14) TOP2A: expression level and patient are proportionate to the susceptibility of Etoposide.
Specific embodiment two
A kind of detection method of using 14 kinds of antitumor medication genes involveds of specimens paraffin embedding slices sample synchronous detection of the present invention, gather specimens and extract nucleic acid, carry out reverse transcription and PCR reaction take patient's nucleic acid as template, finally use electrocapillary phoresis method sample separation, concrete steps are as follows:
1, production is based on the test kit of 14 kinds of antitumor medication genes involveds of use specimens paraffin embedding slices sample synchronous detection of GeXP multiple gene expression genetic analysis systems, and the component that comprises in the test kit is with above-mentioned embodiment 1;
2, collecting sample and extract nucleic acid
Gather the separation and Culture thing of specimens specimens paraffin embedding slices sample, from the separation and Culture thing, extract nucleic acid;
3, carry out reverse transcription (RT) reaction take patient's nucleic acid as template
1) add reagent and sample (the RT plate sees Table 2) in following ratio in 96 hole sample panel:
Table 2RT reaction reagent and sample mix ratio
| The RT reaction reagent | Amount/hole |
| DEPC water (without RNA enzyme/DNA enzyme ultrapure water Dnase/Rnase Free) | 8μL |
| 5 * RT damping fluid | 4μL |
| RT primer solution (each RT primer concentration is 500nM) | 2μL |
| The RT enzyme | 1μL |
| Sample RNA (5-20ng/ul) | 5μL |
| Total | 20μL |
Annotate: add in the RT reaction, positive reference substance is the RNA mixture that extracts from tumour cell, comprises described 14 kinds of antitumor medication related gene mRNAs: consumption is 5 μ L/ reactions
2) hatch (seeing Table 3) by following temperature behind the mixing:
Table 3RT reaction conditions
4, carry out the PCR reaction take reverse transcription product as template
1) add reagent and sample (the PCR plate sees Table 4) in following ratio in 96 hole sample panel:
Table 4PCR reaction reagent and sample mix ratio
| The PCR reaction reagent | Amount/hole |
| 10 * PCR damping fluid | 4μL |
| 25mM?MgCl2 | 4μL |
| The PCR primer | 2μL |
| Archaeal dna polymerase | 1.4μL |
| X solution | 2μL |
| The RT product | 8.6μL |
| Total | 20μL |
Annotate: X solution comprises triphosphate deoxy-nucleotide (dNTPs) and universal primer, universal primer forward amplimer sequence is AGGTGACACTATAGAATA, the reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, universal primer forward amplimer band fluorescent mark.
2) carry out thermal cycle reaction (seeing Table 5) by following temperature behind the mixing:
Table 5PCR reaction conditions
| Step | Temperature | Time |
| 1 | 95°C | 10 minutes |
| 2 | 94°C | 30 seconds |
| 3 | 55°C | 30 seconds |
| 4 | 70°C | 1 minute |
| 5 | N/A | Repeat 2-4 step 34 time (totally 35 times) |
| 6 | 70°C | 1 minute |
| 7 | 4°C | Continue: until collect the PCR product |
5, GeXP genetic analyzer electrocapillary phoresis sample separation
1) preparation GeXP sample (seeing Table 6):
Table 6GeXP sample mix ratio
2) electrocapillary phoresis sample separation
The GeXP sample is added in the hole of proper number on the 96 hole capillary electrophoresis separation plates and carry out capillary electrophoresis separation; Capillary electrophoresis separation is the Novel liquid-phase isolation technique of a class take kapillary as split tunnel, take high-voltage dc as motivating force, and specific procedure is 90 ℃ of sex change 120 seconds, sample introduction voltage 2kv, 30 seconds, separation voltage 6kv, 35 minutes.
6, interpretation of result (seeing GenomeLab GeXP genetic analyzer specification sheets)
According to the parameter of giving tacit consent on the own software of GeXP genetic analyzer the result is carried out the clip size analysis, its X-coordinate represents clip size, and ordinate zou is that signal is strong and weak.According to the peak collection of illustrative plates that the software of GeXP genetic analyzer obtains, the calculated by peak area at each gene peak obtains relative expression's level of antitumor medication genes involved, instructs the nearly medicining condition of 24 kinds of antitumor drugs according to relative expression's level of these genes.Standard diagram as shown in Figure 1, its result can accurately detect the expression level of 14 kinds of antitumor medication genes involveds, each target fragment size interval is moderate, and signal is unlikely to supersaturation, signal is relatively fair between each target, and does not have broad peak, the phenomenon such as bimodal.
Specific embodiment three
Detection kit sensitivity, specificity analyses
Sensitivity analysis: by behind certain ng value doubling dilution, until can't detect signal, this ng value is the lowest detection line, namely the sensitivity of test kit through pcr amplification and capillary electrophoresis detection with positive reference substance.The sensitivity of this test kit is 1.0ng.
Specificity analyses: it is the unimodal of target fragment size that the substance pcr amplification detects through capillary electrophoresis.
Above-mentioned explanation is not limitation of the present invention, and the present invention also is not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement also should belong to protection scope of the present invention.
Claims (4)
1. test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection, comprise DEPC water, 5 * RT damping fluid, the reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, the PCR primer, the 25mM magnesium chloride solution, archaeal dna polymerase and positive reference substance, it is characterized in that described reverse transcription primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds in the following table and the RT amplimer of RNA confidential reference items, described PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds in the following table, forward and reverse pcr amplification primer of the pcr amplification primer of RNA confidential reference items and DNA confidential reference items, gene order is as shown in the table:
2. a kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection according to claim 1, it is characterized in that: described X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; The reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
3. a kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection according to claim 1, it is characterized in that: described positive reference substance comprises described 14 kinds of antitumor medication related gene mRNAs for the RNA mixture for extracting from tumour cell.
4. method of utilizing the antitumor medication related gene expression of the detection level of the test kit of 14 kinds of antitumor medication related gene expression levels of each described use specimens paraffin embedding slices sample synchronous detection among the claim 1-3 is characterized in that specifically may further comprise the steps:
(1) collecting sample and extract nucleic acid
Gather the separation and Culture thing of specimens specimens paraffin embedding slices sample, from the separation and Culture thing, extract nucleic acid;
(2) carry out the RT reaction take patient's nucleic acid as template
Get the nucleic acid samples RNA5 μ L of 5-20ng/ul, DEPC water 8 μ L, 5 * RT damping fluid, 4 μ L, RT primer solution 2 μ L join on the 96 hole sample panel behind the RT enzyme 1 μ L mixing and carry out reverse transcription, reaction conditions: 48 ° C1 minute; 42 ° C60 minute; 95 ° C5 minute; 4 ° of C are until collect the RT product; Wherein each RT primer concentration is 500nM in the RT primer solution, and described RT primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds and the RT amplimer of RNA confidential reference items, and gene order is shown in SEQ ID NO.1～NO.18 in the sequence table;
(3) carry out the PCR reaction take reverse transcription product as template
Get RT product 8.6 μ L, 10 * PCR damping fluid, 2 μ L, the magnesium chloride 4 μ L of 25mM, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L join the reaction of the enterprising performing PCR of 96 hole sample panel, reaction conditions behind the X solution 2 μ L mixings: 95 ° C10 minute; 94 ° of C30 seconds, 55 ° of C30 seconds, 70 ° C1 minute, circulate 35 times; 70 ° C1 minute; 4 ° of C are until collect the PCR product; Wherein said X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; The reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark, each PCR primer concentration is 200nM in the PCR primer solution, the PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds, the pcr amplification primer of RNA confidential reference items and forward and reverse pcr amplification primer of DNA confidential reference items, and gene order is shown in SEQ ID NO.19～NO.38 in the sequence table;
(4) GeXP genetic analyzer electrocapillary phoresis sample separation
Get PCR product 0.1-1 μ L, the sample-loading buffer 38.75 μ L that the GeXP genetic analyzer is supporting, DNA standard substance 0.5 μ L, join after one in mineral oil mixes on the 96 hole parting liquid plates and carry out the electrocapillary phoresis sample separation, the collection of illustrative plates that the software of GeXP genetic analyzer is obtained contrasts with standard diagram, obtains the expression level of antitumor medication genes involved.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310032768.2A CN103305600B (en) | 2013-01-25 | 2013-01-25 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310032768.2A CN103305600B (en) | 2013-01-25 | 2013-01-25 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103305600A true CN103305600A (en) | 2013-09-18 |
| CN103305600B CN103305600B (en) | 2014-09-17 |
Family
ID=49131317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310032768.2A Active CN103305600B (en) | 2013-01-25 | 2013-01-25 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103305600B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105699142A (en) * | 2016-02-18 | 2016-06-22 | 石河子大学 | Paraffin section making method for close-texture hard plant materials |
| CN111394467A (en) * | 2020-04-30 | 2020-07-10 | 浙江树人学院（浙江树人大学） | Detection reagent and method for expression quantity of cancer treatment drug related gene and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851674A (en) * | 2010-01-26 | 2010-10-06 | 中国人民解放军总医院 | Gene expression method for simultaneously detecting 11 sports-related genes and 4 internal reference genes in human blood |
| CN102251036A (en) * | 2011-07-07 | 2011-11-23 | 中国人民解放军总医院 | Method for simultaneously detecting 17 genes |
-
2013
- 2013-01-25 CN CN201310032768.2A patent/CN103305600B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851674A (en) * | 2010-01-26 | 2010-10-06 | 中国人民解放军总医院 | Gene expression method for simultaneously detecting 11 sports-related genes and 4 internal reference genes in human blood |
| CN102251036A (en) * | 2011-07-07 | 2011-11-23 | 中国人民解放军总医院 | Method for simultaneously detecting 17 genes |
Non-Patent Citations (3)
| Title |
|---|
| ALEX J. RAI, ET.AL.,: ""Analytical validation of the GeXP analyzer and design of a workflow for cancer-biomarker discovery using multiplexed gene-expression profiling"", 《ANAL BIOANAL CHEM》 * |
| 胡秀梅;张勇;徐邦牢;杨梦婕;王淼;张晨;李瑾;白如银: ""GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用"", 《病毒学报》 * |
| 董矜;田亚平;张阳东;李立青: ""GeXP多重基因表达系统检测外周炎性相关基因方法学探索"", 《标记免疫分析与临床》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105699142A (en) * | 2016-02-18 | 2016-06-22 | 石河子大学 | Paraffin section making method for close-texture hard plant materials |
| CN105699142B (en) * | 2016-02-18 | 2018-04-03 | 石河子大学 | Paraffin section preparation method for the close hard vegetable material of quality |
| CN111394467A (en) * | 2020-04-30 | 2020-07-10 | 浙江树人学院（浙江树人大学） | Detection reagent and method for expression quantity of cancer treatment drug related gene and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103305600B (en) | 2014-09-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103074434B (en) | CYP2C19 gene polymorphyism detection kit and detection method thereof | |
| CN103074436B (en) | Multi-gene detection kit for guiding administration of 5-fluorouracil and detection method of multi-gene detection kit | |
| CN103667514B (en) | A kind of human interleukin 2 8B gene pleiomorphism fluorescence PCR detection reagent kits | |
| CN103849681B (en) | A kind of Primer composition, multiple gene detection kit and using method thereof instructing medicine of glonoin and health to drink | |
| CN103074435B (en) | Kit for synchronously detecting related gene expression levels of 14 antineoplastic drugs and detection method of kit | |
| CN107227361A (en) | Primer, probe and detection kit for detecting CYP2C19 gene pleiomorphisms | |
| CN105274190A (en) | HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T | |
| CN106086192A (en) | The parting detecting reagent of tacrolimus personalized medicine related gene | |
| CN103074452B (en) | Kit for synchronously detecting fifteen hemorrhagic fever pathogens and detection method of kit | |
| CN104830852A (en) | Multiplex real-time fluorescent PCR (polymerase chain reaction) method for detecting HLA-B*15:02 alleles | |
| CN103305600B (en) | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof | |
| CN103882138A (en) | Multiple-gene detecting kit related to antitumor drugs | |
| CN103074438B (en) | Multi-gene detection kit for guiding administration of warfarin and detection method of multi-gene detection kit | |
| CN103882139B (en) | Multi-gene detection kit for selection of chemotherapy regimens | |
| CN103074437B (en) | Multi-gene detection kit for guiding administration of platinum drugs and detection method of multi-gene detection kit | |
| US20170051332A1 (en) | Methods and compositions for detecting expression of target genes | |
| CN103146841B (en) | Kit capable of synchronously detecting eighteen kinds of fever with eruption pathogens and detection method thereof | |
| CN106701953B (en) | Primer composition and kit for detecting expression level of circadian rhythm control key genes in drosophila melanogaster and using method of primer composition and kit | |
| CN103740831B (en) | Primer combination for guiding application of beta-receptor blocker, multi-gene detection kit and using method thereof | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| CN103215356A (en) | Assay kit for detecting human leukocyte antigen-B (HLA-B)*57:01 and HLA complex P5 (HCP5) alleles | |
| CN102676661B (en) | Fluorescent polarization based homogeneous phase detection method of single nucleotide polymorphism of codon118 of ERCC1 (excision repair cross-complementing 1) gene | |
| CN103725787B (en) | Fluorescent PCR (Polymerase Chain Reaction) detection kit for fusion genes of leukemia | |
| CN102851352A (en) | A neotype real-time fluorescent quantitative detection method for miRNA | |
| CN109652539A (en) | A kind of kit detected for EGFR T790M site mutation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: NINGBO HEALTH GENE TECHNOLOGIES CO., LTD. Free format text: FORMER OWNER: HEALTH BIOMEDICAL CO., LTD. Effective date: 20150422 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 315800 NINGBO, ZHEJIANG PROVINCE TO: 315040 NINGBO, ZHEJIANG PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20150422 Address after: 315040 No. 396 Mingzhu Road, science and Technology Park, Zhejiang, Ningbo Patentee after: NINGBO HEALTH GENE TECHNOLOGIES CO., LTD. Address before: Zhejiang province Ningbo City Creek Street 315800 No. 159 West Village forward half port Patentee before: Health Biological Medicine Co., Ltd. |