CN103205490B - Analyzing method and application of membranous nephropathy tri-methyl status differential expression genes - Google Patents
Analyzing method and application of membranous nephropathy tri-methyl status differential expression genes Download PDFInfo
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Abstract
The invention relates to an analyzing method of membranous nephropathy tri-methyl status differential expression genes. Chromatin immunoprecipitation-high through-put (Chip-seq) sequencing is used for analyzing histone H3K9 tri-methyl status change of peripheral blood mononuclear cells (PBMCs) of a membranous nephropathy patient to obtain the membranous nephropathy tri-methyl status differential expression genes. By intensive researching the differential expression genes, pathogenesis of membranous nephropathy can be further clarified, and new paths are provided for diagnosis and treatment of membranous nephropathy. The analyzing method is reasonable and feasible in design, can effectively help to build a spectrum model of differential expression genes of membranous nephropathy, and related information, as the middle result, of membranous nephropathy can be achieved.
Description
Technical field
The present invention relates to epigenetics research field, especially relate to analytical procedure and the application of the difference expression gene of the tri-methylated state of a kind of membranous nephropathy.
Background technology
Membranous nephropathy is for feature with basilar membrane diffuse thickening and upper subcutaneous immune complex deposit, renal glomerulus also shows unique morphological feature: such as, and basement membrane thickened, particulate state IgG are in glomerular capillary loop deposition or be deposited on subepithelial tissue in electron dense thing.Membranous nephropathy occurs in all over the world, does not have the difference of race, Age and sex.So far, also have many about the pathogenetic research of membranous nephropathy, but still do not find the specific biomarker of membranous nephropathy.Up to now, the histone methylated state of membranous nephropathy changes also unclear.
To the understanding of cell involved in the pathogenesis of membranous nephropathy (MN) and molecule aspect from the research to rat Heymann Nephritis Model, the clinical pathology feature of this model and mankind MN is very similar.A large amount of documents confirms have much about the pathogenetic research of mankind MN, but the specific biological mark of MN does not still find.
Summary of the invention
Based on this, be necessary analytical procedure and the application of the difference expression gene that the tri-methylated state of a kind of membranous nephropathy for studying membranous nephropathy specific biomarkers is provided.
An analytical procedure for the difference expression gene of the tri-methylated state of membranous nephropathy, comprises the steps:
Gather the peripheral blood mononuclear cell of membranous nephropathy patient and normal healthy controls group respectively;
Use the formaldehyde solution of 1% to carry out crosslinking Treatment to described peripheral blood mononuclear cell respectively under room temperature, then wash by PBS solution, then the glycine solution adding 1M stops crosslinked;
Collect the peripheral blood mononuclear cell after being cross-linked respectively, process lysing cell core is carried out with homogenizer, dissolving after collecting lysate is suspended in damping fluid, use H3K9 tri-methylated (H3K9me3) antibody to precipitate lysate in described damping fluid, after the mixture Proteinase K of the DNA obtained and antibody processes in 65 DEG C, obtain the DNA fragmentation of purifying;
Illumina preparation of samples bag is used to carry out end reparation to the DNA fragmentation of described purifying, add joint and clip size selection process, after selecting removing joint, size is that the DNA fragmentation of 100bp checks order, the sequence obtained checking order and the reference genome of bosTau database are compared, allow the mispairing of two bases, deletion can not comparison to reference to genomic sequence, then MACS method is used the sequence analysis after comparison to be determined to the binding site of transcription factor, the DNA enrichment region of ChIP-seq data set and the tri-methylated correspondence of H3K9, use the analytical results of MEME software to MACS method to process and obtain new corresponding DNA die body tri-methylated with H3K9, and find relevant gene tri-methylated to H3K9 according to described DNA die body, and
The tri-methylated relevant gene of described H3K9 of comparative film nephrotic and normal healthy controls group obtains the difference expression gene of the tri-methylated state of described membranous nephropathy.
Wherein in an embodiment, described sequencing procedure uses Solexa/Illumina2G sequenator to check order to the DNA fragmentation that size after removing joint is 100bp.
Wherein in an embodiment, it is use shortly to read the reference genome that aligner Bowtie obtains sequence and bosTau database by checking order and compare that the described reference genome by the check order sequence that obtains and bosTau database is compared.
Wherein in an embodiment, described use MACS method is carried out MACS parameter in analytic process to the sequence after comparison and is: bandwidth is 273; Genome Size is 2.70e+09; Tag size is 49; Model folded data is 10,30; P value is 1.00e-05.
The change of analytical procedure by using chromatin imrnunoprecipitation-high-throughput (ChIP-seq) order-checking to carry out the tri-methylated state of histone H 3 K9 of analyzing film nephrotic peripheral blood mononuclear cell (PBMCs) of the difference expression gene of the tri-methylated state of above-mentioned membranous nephropathy, obtain the difference expression gene of the tri-methylated state of membranous nephropathy, further investigate these difference expression genes and will contribute to illustrating further the pathogeny of membranous nephropathy, for the Diagnosis and Treat of membranous nephropathy provides new way.The analytical procedure of the difference expression gene of the tri-methylated state of above-mentioned membranous nephropathy is reasonable in design feasible, effectively can assist the spectrum model setting up a kind of membranous nephropathy difference expression gene, obtain the relevant information of the membranous nephropathy as intermediate result.
A kind of gene detecting chip, described gene detecting chip is fixed with the difference expression gene of the tri-methylated state of membranous nephropathy that aforesaid method obtains.
Wherein in an embodiment, described difference expression gene is at least one in DGCR6, SNX16, CNTN4, BIRC2 and BIRC3.
By said gene detection chip, intermediate result information can be provided for tentative diagnosis membranous nephropathy, testing process is convenient, do not need to use traditional numerous and diverse detecting step, but whether due to membranous nephropathy a kind of syndrome often, also needing after obtaining these intermediate results just can be diagnosed as in conjunction with other detection data is membranous nephropathy.
Accompanying drawing explanation
Fig. 1 is the distribution of the tri-methylated multiple enrichment region (peaks) of H3K9, and wherein transverse axis represents the length of enrichment region, and the longitudinal axis represents the number of enrichment region;
Fig. 2 is the distribution of enrichment region genes involved in full-length genome;
Fig. 3 is enrichment region genes involved GO analytical results figure, and wherein, transverse axis represents GO item, the gene-ratio that left longitudinal axis representative is relevant to GO, the gene dosage that right longitudinal axis representative is relevant to GO;
Fig. 4 is the schematic diagram of DNA motif, and wherein, transverse axis represents the site of this motif, and the total height of the longitudinal axis reacts the conservative property of this motif, and the height of each base represents the frequency that this base occurs.
Embodiment
Mainly below in conjunction with the drawings and the specific embodiments the analytical procedure of the difference expression gene of the tri-methylated state of membranous nephropathy and application to be further described in detail.
The analytical procedure of the difference expression gene of the tri-methylated state of membranous nephropathy of one embodiment, comprises the steps:
Step S110: the peripheral blood mononuclear cell gathering membranous nephropathy patient and normal healthy controls group respectively.
Step S120: use under room temperature the formaldehyde solution of 1% respectively Cytokines in Peripheral Blood Mononuclear carry out crosslinking Treatment, then wash by PBS solution, then the glycine solution adding 1M stops crosslinked.
Step S130: collect the peripheral blood mononuclear cell after being cross-linked respectively, process lysing cell core is carried out with homogenizer, dissolving after collecting lysate is suspended in damping fluid, use the lysate in H3K9 tri-methylated antibody precipitation buffering liquid, after the mixture Proteinase K of the DNA obtained and antibody processes in 65 DEG C, obtain the DNA fragmentation of purifying.
Step S140: use Illumina preparation of samples bag (Illumina Products) DNA fragmentation to purifying to carry out end reparation, add joint and clip size selection process, after selecting removing joint, size is that the DNA fragmentation of 100bp checks order, the sequence obtained checking order and the reference genome of bosTau database are compared, allow the mispairing of two bases, deletion can not comparison to reference to genomic sequence, then MACS method is used the sequence analysis after comparison to be determined to the binding site of transcription factor, the DNA enrichment region of ChIP-seq data set and the tri-methylated correspondence of H3K9, use the analytical results of MEME software to MACS method to process and obtain new corresponding DNA die body tri-methylated with H3K9, and find relevant gene tri-methylated to H3K9 according to DNA die body.
Wherein, sequencing procedure uses Solexa/Illumina2G sequenator to check order to the DNA fragmentation that size after removing joint is 100bp.Compared by the reference genome of the check order sequence that obtains and bosTau database is use shortly to read the reference genome that aligner Bowtie obtains sequence and bosTau database by checking order and compare.Use MACS method is carried out MACS parameter in analytic process to the sequence after comparison and is: bandwidth is 273; Genome Size (genome size) is 2.70e+09; Tag size (tag size) is 49; Model folded data (model fold) is 10,30; P value (p-value cutoff) is 1.00e-05.
Step S150: the tri-methylated relevant gene of H3K9 of comparative film nephrotic and normal healthy controls group obtains the difference expression gene of the tri-methylated state of membranous nephropathy.
The concrete gene obtaining 5 larger differences in the present embodiment and express is DGCR6, SNX16, CNTN4 and BIRC2 of up-regulated respectively, and the BIRC3 of a down-regulated expression.
The change of analytical procedure by using chromatin imrnunoprecipitation-high-flux sequence (ChIP-seq) to carry out the tri-methylated state of histone H 3 K9 of analyzing film nephrotic peripheral blood mononuclear cell (PBMCs) of the difference expression gene of the tri-methylated state of above-mentioned membranous nephropathy, obtain the difference expression gene of the tri-methylated state of membranous nephropathy, further investigate these difference expression gene levels and will contribute to illustrating further the pathogeny of membranous nephropathy, for the Diagnosis and Treat of membranous nephropathy provides new way.The analytical procedure of the difference expression gene of the tri-methylated state of above-mentioned membranous nephropathy is reasonable in design feasible, effectively can assist the spectrum model setting up a kind of membranous nephropathy difference expression gene, obtain the relevant information of the membranous nephropathy as intermediate result.
In addition, present embodiment additionally provides a kind of gene detecting chip, this gene detecting chip is fixed with the difference expression gene of the tri-methylated state of membranous nephropathy that aforesaid method obtains as detection probes.
Preferably, above-mentioned difference expression gene is at least one in DGCR6, SNX16, CNTN4, BIRC2 and BIRC3.
The expression level of the genes involved of patient to be measured can be detected by said gene detection chip, obtain tentative diagnosis membranous nephropathy and intermediate result information is provided, testing process is convenient, do not need to use traditional numerous and diverse detecting step, but whether due to membranous nephropathy a kind of syndrome often, also needing after obtaining these intermediate results just can be diagnosed as in conjunction with other detection data is membranous nephropathy.
Be below specific embodiment part:
1. materials and methods
The collection of 1.1 samples
The present embodiment has 20 samples, in table 1, wherein, and disease group 10 people, normal healthy controls group 10 people.Disease group patient derives from nephrology of hospital of Guilin City the 181st, is the patient being diagnosed as membranous nephropathy through Renal biospy, and eliminates the interference of inflammation, diabetes and other diseases.Normal healthy controls group and disease group age, race, sex match.The agreement of Hospital Ethical Committee of Guilin City the 181st has been obtained in this research.
The demography of table 1 disease group and control group and Clinical symptoms
Note: numerical value is mean number ± standard deviation
The separation of 1.2 peripheral blood mononuclear cell
Obtain blood sample from disease group and normal healthy controls group, everyone 5ml, dilute with the phosphate buffered saline buffer (PBS damping fluid) of equimultiple volume, join in the lymphocyte separation medium of equimultiple volume after mixing, leave heart 25min at 25 DEG C, 2700.Draw mononuclearcell layer, carry out washing to remove unnecessary blood plasma and parting liquid with PBS damping fluid, then cell sample is kept in-80 DEG C of refrigerators for subsequent use.
1.3 chromatin imrnunoprecipitation
The process of the present embodiment to traditional dyeing matter immunoprecipitation (ChIP) is changed, specific as follows: be cross-linked cell by the formaldehyde solution of 1% under room temperature, time 10min; Wash with cold PBS damping fluid subsequently, then the glycine solution adding 1M stops crosslinked.Collect crosslinked cell, with homogenizer, process is carried out to crosslinked cell and make it lysing cell core, collect lysate, dissolve and be suspended in damping fluid; Re-use H3K9 tri-methylated antibody resolution of precipitate thing, 4 DEG C are spent the night.Immunocomplex Proteinase K, in 65 DEG C of process 2h, carries out purifying to DNA.
1.4 high-flux sequence
Prepare the standard program of Illumina order-checking, order-checking and Quality Control to provide by Illumina company, specific as follows: first to use Illumina preparation of samples bag to process DNA fragmentation, comprise end reparation, add the selection of joint and clip size, then constructed dna library, carries out the reading of high-throughout order-checking and data.
1.5ChIP-seq interpretation of result
Agarose gel electrophoresis process is carried out to the DNA sheet of above-mentioned process, therefrom isolates the fragment that size is 100bp (not containing joint), and use Solexa/Illumina2G sequenator to check order.Then to utilize rapidly and efficiently short reads, on reference genome that aligner (Bowtie sequenator) issues sequence label comparison to bosTau2007, to allow the mispairing of two bases.Utilize MACS method to carry out Analysis and Identification to the sequence after comparison and go out enrichment region (peak).MACS is one and simply and effectively analyzes Eukaryotic technology, and its object determines Binding site for transcription factor exactly, the data set of ChIP-seq and the enrichment region of histone modification.During analysis, can not uniquely comparison to can be deleted with reference to genomic sequence.MACS parameter is: bandwidth=273; Genome size=2.70e+09; Tag size=49; Model fold=10,30; P-value cutoff=1.00e-05.Use AmiGO(version 1.8) multiple enrichment region (peaks) is contrasted to reference on genome.AmiGO is a web application, and user can carry out searching classifiably, arrange, analyze and the data notes of genes involved product.The data that the website visiting GO association that AmiGO can enter Gene Ontology (GO) provides.Motif analyzes and uses MEME4.7.0 software.
2 results
2.1 chromatin imrnunoprecipitation-high-throughput (ChIP-seq) checks order
Use the chromatin fragments of the peripheral blood mononuclear cell of the tri-methylated specific antibody precipitation normal healthy controls group of H3K9 and disease group, form immunocomplex precipitation.Utilize Bowtie by the sequence alignment that records to reference to genome, comparison result is in table 2.All ChIP-seq data can well comparison to reference to genome, have 12899881 reads and 12440646(96.44%) reads of bar comparison, cover 539358043 bases.
Table 2 ChIP-seq sequence alignment result is added up
Note: comparison reads number: can comparison to reference to genomic reads number;
Unique comparison reads number: energy comparison is to the reads number with reference to genome unique positions;
Comparison rate: the ratio of comparison reads number and total reads number;
Unique comparison rate: the ratio of unique comparison reads number and total reads number.
The tri-methylated distribution at full-length genome of 2.2 membranous nephropathy patient mononuclearcell (PBMCs) H3K9
By the enrichment region using MACS to identify histone modification, the scope calculating district is 5000 bases.ChIP-seq records raw 217 enrichment regions of membranous nephropathy group common property, and total length is 192047bp.The distribution of enrichment region be typical priority allocation general layout as depicted in figs. 1 and 2, the about 150-200bp of the distance between enrichment region.
2.3 enrichment region genes involved screenings are analyzed with GO functional clustering
Enrichment region (peak district) location on genome combined with target protein (tri-methylated H3K9) is relevant with which gene, can reflect target protein or the protein modified target gene district that may regulate and control of particular group to a certain extent.In order to understand the function of enrichment region genes involved further, use the analysis of GO functional clustering.The analysis of GO functional clustering is the categorizing system of a kind of conformability, unitized, Open Dynamic real-time update, it comprises three large independently bodies: the vital process (biological process) that gene participates in, the Molecular biological function (molecular function) of residing cellular component and element (cellular component) and performance.Independently can go out again different subgrade time below three bodies, form the tree-shaped branched structure of a body layer by layer downwards.By analyzing the GO functional clustering of enrichment region, can know that enrichment region genes involved relates to the change of which biological function.
In the present embodiment, Molecular biological function (molecular function) this three major types that 86 genes relate to the vital process (biological process) of gene participation, residing cellular component and element (cellular component) and play is learnt by the analysis of GO functional clustering, as shown in Figure 3, in cellular component and this class of element, have 28 gene annotation to cellular portions, 14 relate to organoid.Relate to combination in Molecular biological function and the gene of catalytic activity is 25 and 12 respectively, regulatory enzyme activity account for 13.3%.This class of vital process participated in, have 13 genes to participate in metabolic process, 10 relate to regulatory function, and 5 relate to component organization function.
The die body (DNA motif) of 2.4 enrichment region genes involveds is analyzed
By the function DNA motif that high-throughput functional genomics technology for detection arrives, these data can provide direct or indirect evidence for the adjustment of gene.MEME is one of effective tool finding new motif in large-scale genomic sequence data, and as E value < 1.0e-05, motif is believable.Utilize MEME(edition 4 .7.0) from the data that ChIP-seq records, have found 5 motif, as shown in table 3 and fig. 4.New H3K9 shows in conjunction with the discovery of motif, and the histone modification of different loci changes the preference of the histone H 3 combined based on sequence.
Table 3.motif essential information
Note: fraction of coverage refers to the fraction of coverage of this motif on remaining peak
In table, the base sequence of motif represents with degeneracy base, and the concrete meaning of each base is as follows:
S:C+G, namely this site may be C base or G base;
R:A+G, namely this site may be A base or G base;
W:A+T, namely this site may be A base or T base;
K:T+G, namely this site may be T base or G base;
Y:C+T: namely this site may be C base or T base.
The contrast of 2.5 disease group and the tri-methylated state of control group H3K9
By the above-mentioned analysis to sequence results also compared with normal healthy controls group, 108 genes relating to the tri-methylated state of H3K9 are had to show great difference in membranous nephropathy patient.In the tri-methylated differential gene of these H3K9, have 75 rises, 33 performance down function, pick 15 genes differed greatly in table 4 in table 4.Wherein, DGCR6, SNX16, CNTN4, BIRC2 and BIRC35 gene presents maximum difference, in close relations with membranous nephropathy.
15 that select in the table 4.ChIP-seq result genes showing larger difference in the tri-methylated state of membranous nephropathy patient H3K9
In table 4, the miRNA of these differential expressions can as the probe molecule of gene detecting chip, hybridized by the DNA molecular extracted with peripheral blood mononuclear cells to be measured, the expression level of the relevant DNA of patient to be measured is judged again by follow-up colour developing etc., particularly above-mentioned differential expression DNA, can, as the intermediate result of diagnosis membranous nephropathy, whether be membranous nephropathy in conjunction with the diagnosis of other detected results.
3 discussion of results
Histone is end modified be a kind of special can the regulatory gene codon of expressing.Histone H 3 K9 is tri-methylated belongs to posttranslational modification, main relevant with Transcription inhibition with heterochromatic formation.The present embodiment, by using ChIP-seq, a kind ofly can identify the binding site of transcription factor and the technology of other protein be combined with DNA within the scope of full-length genome.Obtain the tri-methylated relevant difference expression gene of membranous nephropathy, for the pathogenesis studying membranous nephropathy further provides help.This embodiment Main Analysis tri-methylated state of H3K9 of membranous nephropathy patient and healthy person.These methylation state significant differences candidate gene synthesize with immunity, cell signalling, Protein transport, passage and transport and extracellular matrix etc. relevant.
In candidate gene, DGCR6 up-regulated.DGCR6 is positioned at No. 22 karyomit(e) q11, has the copy (DGCR6 and DGCR6L) of two very high homology, and its disappearance causes DiGeorge syndrome.The most possible biochemical activity of the product of this gene is exactly the regulation and control to gene.In heart, kidney, suprarenal gland, all there is high-caliber DGCR6 protein, also exist in liver and spleen.
The sorting of SNX16 coding connects a member of protein family, and this family all comprises a PHOX(PX) territory.PX territory is a Phosphoinositides-binding domains, is a kind of new PI combination, approximately adjustable 47 Mammals film positioning proteins, wherein 30 to connect protein family (SNXs) relevant with sorting.There are some SNXs in endocytic pathway, participate in the adjustment of film transhipment.SNX16 is a kind of novel protein containing 343 amino-acid residues, after comprising a PX territory, a coiled-coil domain and C-end region.The same with other sorting protein, SNX16 contacts by producing with the interactional PX territory of lecithin phospholipid acyl inositol-3-phosphoric acid and film.The function in PX territory is that PX territory may participate in protein-protein interaction, and many PX territories comprise abundant PxxP motif, shows that they may mediate the interaction with SH3 structural domain for the protein containing abundant phosphatidylinositols (3) P or other PI films.PX territory also may regulate the activity of its host protein.
A member of CNTN4 coding attachment proteins family, belongs to the member of immunoglobulin class.In the present embodiment, research finds that CNTN4 genetic expression is raised, and this may be relevant with membranous nephropathy immune complex deposit.The deposition of IgG1, IgG2, IgG3 and IgG4 can be detected in membranous nephropathy patient.Attachment proteins is the cell adhesion molecule be connected with aixs cylinder, works in the formation and plasticity-of neural network.The albumen of CNTN4 coding is a kind of neuron membrane albumen of glycosyl grappling, plays a role to the formation that aixs cylinder in nervous system development process connects.Disappearance or the sudden change of this gene can cause the neurodevelopmental disorder comprising autism.CNTN4 and 16 type cerebellar ataxia diseases are related, and the disappearance of the CNTN4 of a single copy is just enough to cause the 3p comprising hypoevolutism to lack syndromes.Therefore, CNTN4 is one of few in number autosomal gene be identified, and these genes are relevant with cognitive defect in continuous print gene syndrome.
BIRC2 and BIRC3 encodes a member of IAP family, by conjunction with TNF (tumor necrosis factor) receptor associated factor TRAF1 and TRAF2 inhibited apoptosis.TRAF2 is the signal that main connection albumen is responsible for being caused by TNFR2, and its degraded can regulate the biological activity of this acceptor.The apoptosis that coded protein can suppress serum deprivation to be induced, but do not affect the apoptosis caused because being exposed to vitamin k4.TRAF2 albumen comprises 3 IAP to be repeated and a ring finger.The death of IAP protein on cells plays down regulation; Cell survival is played a significant role, mainly by stoping the transduction of apoptosis, conditioning signal and regulating cell propagation to realize.BIRC2 has vital effect in the existence maintaining endotheliocyte and vessel homeostasis.BIRC2 regulates the survival of endotheliocyte by the signal that regulation and control TRAF2 relies on, or by the ubiquitination target protein in TNFR-related complex, as NEMO and TRAF221.BIRC2, by regulating the signal of endothelial cell death acceptor, or by regulating the formation of Tumor Necrosis Factor Receptors mixture to play a role.BIRC3 promotes the survival of estrogen-dependent breast cancer cells and stops in the necrocytosis of TNF α induction to play an important role.The character of BIRC3ERE is almost identical with a consistent sequence, and is converted to one and do not show the perfect concensus sequence increasing ER activity, and this shows that another factor may stop the interaction of ER and this binding site.
In sum, the tri-methylated state of H3K9 of system evaluation membranous nephropathy peripheral blood mononuclear cells, obtain between histone methylated and membranous nephropathy and contact, the tri-methylated non-physiologic environment relating to membranous nephropathy of H3K9, and these new candidate genes may become potential biomarker or therapy target.The effect of the tri-methylated candidate gene of H3K9 in the pathogenesis of membranous nephropathy can be specified by studying further.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. an analytical procedure for the difference expression gene of the tri-methylated state of the membranous nephropathy of non-diseases diagnoses and treatment object, is characterized in that, comprise the steps:
Gather the peripheral blood mononuclear cell of membranous nephropathy patient and normal healthy controls group respectively;
Use the formaldehyde solution of 1% to carry out crosslinking Treatment to described peripheral blood mononuclear cell respectively under room temperature, then wash by PBS solution, then the glycine solution adding 1M stops crosslinked;
Collect the peripheral blood mononuclear cell after being cross-linked respectively, process lysing cell core is carried out with homogenizer, dissolving after collecting lysate is suspended in damping fluid, use H3K9 tri-methylated antibody to precipitate lysate in described damping fluid, after the mixture Proteinase K of the DNA obtained and antibody processes in 65 DEG C, obtain the DNA fragmentation of purifying;
Illumina preparation of samples bag is used to carry out end reparation to the DNA fragmentation of described purifying, add joint and clip size selection process, after selecting removing joint, size is that the DNA fragmentation of 100bp checks order, the sequence obtained checking order and the reference genome of bosTau database are compared, allow the mispairing of two bases, deletion can not comparison to reference to genomic sequence, then MACS method is used the sequence analysis after comparison to be determined to the binding site of transcription factor, the DNA enrichment region of ChIP-seq data set and the tri-methylated correspondence of H3K9, use the analytical results of MEME software to MACS method to process and obtain new corresponding DNA die body tri-methylated with H3K9, and find relevant gene tri-methylated to H3K9 according to described DNA die body, and
The tri-methylated relevant gene of described and H3K9 of comparative film nephrotic and normal healthy controls group obtains the difference expression gene of the tri-methylated state of described membranous nephropathy.
2. the analytical procedure of the difference expression gene of the tri-methylated state of membranous nephropathy of non-diseases diagnoses and treatment object as claimed in claim 1, it is characterized in that, described sequencing procedure uses Solexa/Illumina2G sequenator to check order to the DNA fragmentation that size after removing joint is 100bp.
3. the analytical procedure of the difference expression gene of the tri-methylated state of membranous nephropathy of non-diseases diagnoses and treatment object as claimed in claim 1, it is characterized in that, it is use shortly to read the reference genome that aligner Bowtie obtains sequence and bosTau database by checking order and compare that the described reference genome by the check order sequence that obtains and bosTau database is compared.
4. the analytical procedure of the difference expression gene of the tri-methylated state of membranous nephropathy of non-diseases diagnoses and treatment object as claimed in claim 1, it is characterized in that, described use MACS method is carried out MACS parameter in analytic process to the sequence after comparison and is: bandwidth is 273; Genome Size is 2.70e+09; Tag size is 49; Model folded data is 10,30; P value is 1.00e-05.
5. difference expression gene DGCR6, SNX16, CNTN4, BIRC2 and BIRC3 application in the detection chip of the tri-methylated state of preparation diagnosis membranous nephropathy of the tri-methylated state of the membranous nephropathy that the method according to any one of Claims 1 to 4 obtains.
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| CN103205490A (en) | 2013-07-17 |
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