甲基化抑制剂 induce expression of the human reelin and glutamic acid
DNA Methyltransferase Inhibitors Coordinately Induce Expression of the Human Reelin and Glutamic Acid Decarboxylase 67Genes
Marija Kundakovic,Ying Chen,Erminio Costa,and Dennis R.Grayson
The Psychiatric Institute,Department of Psychiatry,College of Medicine,University of Illinois at Chicago,Chicago,Illinois
Received September 6,2006;accepted October 24,2006
ABSTRACT
Reelin and glutamic acid decarboxylase 67(GAD67)mRNAs and protein levels are substantially reduced in postmortem brains of patients with schizophrenia.Increasing evidence sug-gests that the observed down-regulation of reelin and GAD67gene expression may be caused by dysfunction of the epige-netic regulatory mechanisms operative in cortical GABAergic interneurons.To explore whether human reelin and GAD67mRNAs are coordinately regulated through DNA methylation-dependent mechanisms,we studied the effects of DNA meth-yltransferase inhibitors on reelin and GAD67expression in NT-2neuronal precursor https://www.sodocs.net/doc/97209073.html,petitive reverse transcription-polymerase chain reaction with internal standards was used to quantitate mRNA levels.The data showed that reelin and GAD67mRNAs are induced in the same dose-and time-de-pendent manners.We further demonstrated that the activation of these two genes correlated with a reduction in DNA methyl-
transferase activity and DNA methyltransferase 1(DNMT1)pro-tein levels.Time course Western blot analysis showed that DNMT1protein down-regulation occurs temporally before the reelin and GAD67mRNA increase.In addition,chromatin im-munoprecipitation assays demonstrated that the activation of the reelin gene correlates with the dissociation of DNMT1and methyl-CpG binding protein 2(MeCP2)from the promoter,and an increased acetylation of histones H3in the region.Together,our data strongly imply that human reelin and GAD67genes are coordinately regulated through epigenetic mechanisms that in-clude the action of DNMT1.Our study also suggests that neg-ative regulation of the reelin gene involves methylation-depen-dent recruitment of DNMT1,MeCP2,and certain histone deacetylases,which most likely reduce the activity of the pro-moter by shifting the surrounding chromatin into a more com-pact state.
It is now well established that disruption of epigenetic mechanisms can give rise to a variety of disorders in humans,including some that are associated with cognitive abnormal-ities (Egger et al.,2004;Jiang et al.,2004;Levenson and Sweatt,2005).For instance,mutations in the methyl-CpG binding protein MeCP2are responsible for 90to 95%of all cases of Rett syndrome,one of the most common causes of mental retardation in the female population (Weaving et al.,2005).These findings,together with studies using condi-tional DNA methyltransferase 1(DNMT1)mutant mice,sug-gest that DNA methylation is essential for proper neuronal function (Fan et al.,2001;Tucker,2001).It seems that DNA methylation may be an important mechanism associated
with the dynamic regulation of genes expressed in neurons,especially those involved in synaptic plasticity,such as reelin and brain-derived neurotrophic factor (Martinowich et al.,2003;Levenson et al.,2006).
Increasing evidence indicates that the dysfunctions seen in schizophrenia may be caused by an epigenetically induced down-regulation of GABAergic neuronal markers,such as reelin and glutamic acid decarboxylase 67(GAD67)(Costa et al.,2004;Guidotti et al.,2005).In adult brain,reelin most likely plays an important role in synaptic plasticity,learning,and memory formation (Qiu et al.,2006).GAD67is one of the two key enzymes involved in the synthesis of GABA (Guidotti et al.,2005).The decreases in reelin and GAD67mRNA and protein levels are among the most consistently replicated findings reported in postmortem brains of patients with schizophrenia (Fatemi et al.,2000;Guidotti et al.,2000;Eastwood and Harrison,2003;Torrey et al.,2005).A recent study demonstrated that the same GABAergic neurons that
This work was supported by grant MH62682-05from the National Insti-tutes of Mental Health (to D.R.G.).
Article,publication date,and citation information can be found at https://www.sodocs.net/doc/97209073.html,.doi:10.1124/mol.106.030635.ABBREVIATIONS:DNMT,DNA methyltransferase;AZA,5-aza-2?-deoxycytidine;ChIP,chromatin immunoprecipitation;DOXO,doxorubicin;G3PDH,glyceraldehyde 3-phosphate dehydrogenase;GAD67,glutamic acid decarboxylase 67;HDAC,histone deacetylase;MeCP2,methyl-CpG binding protein 2;NT-2,N-tera 2neuronal progenitor cells;ZEB,zebularine;ANOVA,analysis of variance;PCR,polymerase chain reaction;RT-PCR,reverse transcription-polymerase chain reaction;bp,base pair(s);PBS,phosphate-buffered saline.0026-895X/07/7103-644–653$20.00M OLECULAR P HARMACOLOGY
Vol.71,No.3Copyright ?2007The American Society for Pharmacology and Experimental Therapeutics 30635/3166907Mol Pharmacol 71:644–653,2007
Printed in U.S.A.
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express reelin and GAD67exhibit an up-regulation of the mRNA that encodes DNMT1(Veldic et al.,2004).We and others have also shown that the reelin promoter is hyper-methylated in the brains of patients with schizophrenia com-pared with control subjects (Abdolmaleky et al.,2005;Gray-son et al.,2005).Together,these findings support our hypothesis that down-regulation of reelin,GAD67,and prob-ably other mRNAs and proteins expressed in GABAergic neurons may be caused by mechanisms mediated through DNMT1-induced hypermethylation of the corresponding CpG island-containing promoters (Grayson et al.,2006).We have already accumulated evidence showing that the human reelin gene is epigenetically regulated through changes in the methylation status of the https://www.sodocs.net/doc/97209073.html,ing NT-2neuronal precursor cells,we have shown that the reelin promoter is more heavily methylated when the gene is silent (Chen et al.,2002).Activation of the reelin gene by various agents,including retinoic acid,the DNA methylation inhibitor 5-aza-2?-deoxycytidine (AZA),and histone deacetylase (HDAC)inhibitors valproic acid and trichostatin A,corresponds with a decrease in promoter methylation.In addition,induction of reelin expression is accompanied by alterations that suggest a more open chro-matin structure.These changes include the appearance of DNase I hypersensitive sites and increased levels of acetyl histone H3and acetyl histone H4histones in the vicinity of the reelin promoter (Chen et al.,2002;Mitchell et al.,2005).
Studies in mice indicate that reelin and GAD67RNAs may be coordinately regulated.Treatment with L -methionine,a precursor of the methyl donor S -adenosyl-methionine (SAM),induced the down-regulation of reelin and GAD67mRNAs and proteins in vivo (Tremolizzo et al.,2002)and in primary neuronal cell cultures in vitro (Noh et al.,2005).This effect of methionine was attenuated by cotransfection of DNMT1an-tisense oligonucleotides,providing a link between the expres-sion of DNMT1and the regulation of reelin and GAD67genes (Noh et al.,2005).In addition,methionine treatment also induced an increased association of the methyl CpG-binding protein MeCP2to mouse reelin and GAD67promoters (Dong et al.,2005).
The aim of the current study was to evaluate the hypoth-esis that the human reelin and GAD67genes are coordi-nately regulated by DNA methylation through the action of DNMT1.To address this,we used neuronal precursor cells (NT-2)and treatments with three distinct DNA methyltrans-ferase inhibitors.Doxorubicin (DOXO)has recently been shown to act as a potent inhibitor of DNMT1activity,most likely acting through DNA intercalation (Yokochi and Rob-ertson,2004).AZA and zebularine (ZEB)are nucleoside an-alogs that after incorporation into replicating DNA form co-valent bonds with DNA methyltransferases and inhibit their function (Egger et al.,2004).Our study strongly suggests that inhibition of DNA methylation and/or DNMT1protein down-regulation lead(s)to coordinate reactivation of human reelin and GAD67gene expression.This study also provides evidence that transcription of the human reelin gene is repressed by the methylation-mediated recruitment of DNMT1,MeCP2,and possibly other corepressors,including certain HDACs.
Materials and Methods
Cell Culture and Drug Treatments.NT-2cells (Stratagene,La Jolla,CA)were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12medium supplemented with 10%fetal bovine serum and 1%penicillin,1%streptomycin,and 1%L -glutamine.DOXO,AZA,and ZEB were obtained from Sigma-Aldrich (St.Louis,MO).Stock solutions of the drugs were prepared by dissolving the substances in either distilled water (DOXO),50%acetic acid (AZA),or dimethyl sulfoxide (ZEB),and stored at ?20°C.For all experiments,control measurements were obtained from vehicle-treated cells.For dose-response quantitative RT-PCR experiments,cells were treated for 48h with the following concentrations of DOXO;10,25,50,100,250,and 1000nM.Time course mRNA experiments and time course Western blot analyses were carried out after cells had been treated with 100nM DOXO for 0,3,6,12,24,36,or 48h.For DNA methyltransferase assays and dose-response Western blot analysis,cultures were either untreated or treated with 100or 250nM DOXO for 48h,whereas for cell viability assays,cells were additionally treated with 2?M DOXO for 48h.Chromatin immunoprecipitation assays and nonquantitative RT-PCR assay for GAD65was per-formed with vehicle-treated cells and cells treated with 250nM DOXO for 48h.For both quantitative RT-PCR experiments and Western blot analysis,cells were treated with 5?M AZA for 48h and with 500?M ZEB for either 48h or 48h followed by 48-h incubation with untreated medium.
Nuclear Extracts.Nuclear extracts of untreated and treated NT-2cells were obtained using NE-PER Nuclear and Cytoplasmic Extraction kit as recommended by the manufacturer (Pierce Biotech-nology,Rockford,IL).The protein concentrations in the extracts were determined using Bio-Rad Protein Assay (Bio-Rad Laborato-ries,Hercules,CA).
Quantitative RT-PCR Analysis.RNA was isolated after ultra-centrifugation through CsCl (Chen et al.,2002).Reelin,GAD67,G3PDH,and DNMT1mRNA contents were measured by competitive RT-PCR with internal standards as described previously (Grayson and Ikonomovic,1999).Primers were designed to minimally cross at least one exon/intron boundary.For example,the 5?DNMT1primer resides in exon 23,whereas the 3?primer was taken from exon 27(Ramchandani et al.,1998).For measuring reelin mRNA (GenBank accession number NM_005045),the internal standard was generated by deleting 160bp in the middle of the 674amplicon using overlap-extension PCR (Auta et al.,2006).PCR was conducted using the forward primer (?2344)5?-ATCCGTGGTGCTGAAGTCAGCTTT-3?and the reverse primer (?3018)5?-TGAGTACTCCAGCTTCACCTG-GTT-3?(annealing temperature ?68°C,30cycles).For GAD67mRNA (GenBank accession number M81883),the internal standard was generated by deleting 74bp of the 414-bp amplicon,and the following primers were used for PCR:the forward primer (?1855),5?-CTTCCAGCCAGACAAGCAGTATGA-3?;and the reverse primer (?2269),5?-TGGGTTGGAGATGACCATCCGGAA-3?(annealing temperature ?60°C,30cycles).For G3PDH (GenBank accession number BC083511),the internal standard was generated by deleting 216bp of the 683-bp amplicon,and PCR was carried out using the forward primer (?237)5?-CTGAGAACGGGAAGCTTGTCATCA-3?and reverse primer (?920)5?-TGTCGCTGTTGAAGTCAGAG-GAGA-3?(annealing temperature ?60°C,30cycles).For measuring DNMT1mRNA (GenBank accession number BC092517),the inter-nal standard was generated by deleting 196bp of the 509-bp ampli-con,and PCR was carried out using the forward primer (?2228)5?-AATCGCATCTCTTGGGTCGGAGAA-3?and the reverse primer (?2737)5?-ACGGGCACAGCTCACACAGAATTT-3?(annealing tem-perature ?65°C,30cycles).The following primers were used for the PCR amplification of GAD65cDNA (GenBank accession number NM_000818):forward primer,(?989)5?-TTTCTCTCAAGAAGG-GAGCTGCAG-3?;and reverse primer (?1788)5?-GGGTTGG-TAGCTGACCATTGTGG-3?(annealing temperature ?60°C,34cycles).
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DNMT Assay.To measure DNA methyl transferase activity,we used a modification of a previously published method (Szyf et al.,1991).A typical methylation reaction (30?l)contained 1?g of oligonucleotides [poly(dI-dC)?poly(dI-dC)](GE Healthcare,Little Chalfont,Buckinghamshire,UK),an appropriate volume of nuclear extract containing 13?g of protein and 12.2nM S -adenosyl-L -[meth-yl-3H]methionine (specific activity,82Ci/mmol;GE Healthcare)in reaction buffer (20mM Tris,pH 7.6,25%glycerol,10mM EDTA,28mM 2-mercaptoethanol,and 0.2mM phenylmethylsulfonyl fluoride).The reaction mixtures were incubated at 37°C for 3h,followed by incubation at 65°C for 10min.Afterward,1ml of 10%trichloroacetic acid was added,and samples were incubated overnight at 4°C.Mix-tures were then filtered through Whatman GF/C glass microfiber filters and washed twice with 2ml of trichloroacetic acid.Filters were immersed in 3ml of scintillation cocktail (Scintiverse;Fisher Scientific,Pittsburgh,PA)for radioactivity counting.
Western Blots.Nuclear extract proteins were separated onto 4to 20%(DNMT1)or 10to 20%(MeCP2)Tris-glycine gels and trans-ferred overnight (DNMT1)or for 2h (MeCP2)to nitrocellulose mem-branes (Invitrogen,Carlsbad,CA).The membranes were blocked with PBS/Tween 20(0.1%)containing 5%nonfat dry milk for 1h followed by an overnight incubation at 4°C with DNMT1polyclonal antibody (1:1000dilution;New England Biolabs,Ipswich,MA)or MeCP2polyclonal antibody (1:500;Abcam,Cambridge,MA).Mem-branes were then rinsed three times in PBS and incubated with peroxidase-labeled secondary antibody (1:3000;GE Healthcare).Im-munoreactive bands were visualized using the enhanced chemilumi-nescence plus Western blotting detection system (GE Healthcare Bio-Sciences).The intensity of ?-actin immunofluorescence was de-termined on the same blots using ?-actin monoclonal antibodies (1:5000dilution;Sigma-Aldrich),and the corresponding signals were used for a comparative estimation of the amounts of protein applied to the gels.Blots were scanned,and bands were visualized using a Storm 860PhosphorImager (GE Healthcare).Band intensities were analyzed using ImageQuant software (GE Healthcare).
Chromatin Immunoprecipitation Assays.Chromatin immu-noprecipitation (ChIP)assays were performed using the ChIP assay kit protocol (Upstate Biotechnology,Lake Placid,NY)as described previously (Mitchell et al.,2005).In brief,107nontreated or DOXO-treated cells were fixed using 1%formaldehyde at room temperature for 10min.Cells were washed twice in ice-cold PBS,resuspended in SDS-lysis buffer,and sonicated until cross-linked chromatin was sheared to an average DNA fragment length of 200to 800bp.The sonicated lysate (5%)was used to quantitate the total amount of DNA present in different samples before immunoprecipitation (in-puts).Chromatin preparations were immunoprecipitated using anti-DNMT1monoclonal antibody (Imgenex,San Diego,CA),anti-MeCP2and anti-acetyl-histone H3polyclonal antibodies (Upstate).Nonimmunoprecipitated samples were used as negative controls.Precipitated complexes were bound to protein G-agarose,washed,and then eluted in 1%SDS/0.1M NaHCO 3.Cross-linking between DNA and proteins was reversed by heating the samples at 65°C overnight,followed by Proteinase K digestion at 65°C for 1h.DNA was recovered by phenol/chloroform extraction and ethanol precipi-tation,and 4?l of a 20-?l sample was analyzed by PCR.The primers for the reelin promoter region were 5?-CCGGGACACGTGTGGCG-GCG-3?(forward,?220bp)and 5?-AAAGCGGGGGTAATAGC-CAGCCGC-3?(reverse,?262bp).The protocol included an initial denaturation cycle (5min,94°C),40cycles of denaturation (1min,94°C),annealing (1min,62°C),and extension (1min,72°C),followed by the final extension cycle (7min,72°C).For the ?-globin locus control region,the forward primer (?3961bp)was 5?-AGACACTT-GCTCTTTCCAGGACTT-3?,whereas the reverse primer (?4250bp)was 5?-TGCCAGTATATGTGCTTCGATAGG-3?.The amplification included an initial denaturation cycle (5min,94°C),40cycles of denaturation (1min,94°C),annealing (1min,55°C),and extension (1min,72°C),followed by the final extension cycle (7min,72°C).PCR amplification products were separated on 1.6%agarose gels,
and optical density readings were determined using a computer-assisted densitometry program (Kodak EDAS 290;Eastman Kodak Co.,Rochester,NY).For all experiments,input and immunoprecipi-tated DNA samples were below saturation levels after PCR.
Cell Viability Assays.Cell cultures were treated with vehicle-containing medium or medium supplemented with 100nM,250nM,or 2?M DOXO.After 48h,medium was removed and replaced with control medium containing 50?M propidium iodide (a marker of cell damage)and 1?M calcein acetoxymethyl ester (a marker of cell viability).After 10min of incubation,the fluorochrome-containing medium was removed and replaced with control medium,and cell density and viability were examined by fluorescence microscopy.Statistical Analyses.All experimental results are expressed as mean ?S.E.M.of three independent experiments (a minimum of three separate measurements were obtained per experiment).Stu-dent’s t test (for ChIP results)and one-way ANOVA followed by the Bonferroni multiple comparison test (for all other results)were used to assess significance of the differences between groups.Analyses were conducted using SigmaStat software (Systat Software,Point Richmond,CA).In addition,dose-response curves for reelin and GAD67gene induction were obtained using Prism version 4(Graph-Pad Software,San Diego,CA).
Results
DOXO Increases Reelin and GAD67Gene Expression in a Similar Dose-and Time-Dependent Manner.The initial step in our study was to explore whether treatment of human neural progenitor (NT-2)cells with DOXO,a drug that acts as a DNMT1inhibitor,would lead to changes in reelin and GAD67mRNA expression.In NT-2cells,back-ground levels of reelin mRNA are barely detectable (0.010?0.0015pg of reelin mRNA per microgram of total RNA),whereas these cells show significant expression of GAD67mRNA (0.12?0.013pg of GAD67mRNA per microgram of total RNA).We observed a dose-dependent increase in both reelin and GAD67mRNA levels after 48h of DOXO treat-ment (Fig.1,A and B).The data showed that expression of the reelin mRNA increased up to 92-fold,at which a maximal response was achieved using 250nM DOXO (0.92?0.15pg of reelin mRNA per microgram of total RNA).Likewise,250nM DOXO led to a 20-fold (maximal)induction of GAD67mRNA (2.5?0.23pg of GAD67mRNA per microgram of total RNA).To demonstrate the specificity of the reelin and GAD67mRNA induction by DOXO,we examined the expres-sion of two additional genes,G3PDH and GAD65,after the same treatment.In contrast to the changes in reelin and GAD67mRNAs,G3PDH mRNA levels were not significantly changed independent of the concentration of drug used (Fig.1,A and B).Likewise,GAD65mRNA,which is not expressed in NT-2cells at readily detectable levels,was not induced even with the DOXO treatment that maximally activated reelin and GAD67mRNAs (Fig.1D).We next constructed dose-response curves to compare EC 50values for reelin and GAD67mRNA induction in response to DOXO.These anal-yses revealed the EC 50values to be 102and 103nM for reelin and GAD67mRNA increases,respectively (Fig.1C).Both the maximal induction at 250nM concentration of the drug to-gether with the nearly equal EC 50values for these mRNAs,suggest that reelin and GAD67are activated in a similar dose-dependent manner.
To explore the time frame in which changes in reelin and GAD67mRNA expression occur,NT-2cells were treated with 100nM DOXO (EC 50value)for various lengths of time.Data
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from these experiments showed that both reelin and GAD67 mRNA levels were increased in a similar temporal manner as well(Fig.2,A and B).Most importantly,the initial induction of both mRNAs occurred12h after initiating drug treatment (Fig.2B).As measured by competitive RT-PCR,incubation of NT-2cells with100nM DOXO for12h increased reelin mRNA levels5.3-fold(from0.019?0.003to0.1?0.002pg of reelin mRNA per microgram of total RNA)and GAD67 mRNA levels4-fold(from0.15?0.015to0.63?0.057pg of GAD67mRNA per microgram of total RNA).Together with the dose-response study,these results suggest that DOXO treatment leads to a coordinated up-regulation of reelin and GAD67mRNA expression.
Induction of Reelin and GAD67Genes Is Associated with Reduced DNMT Enzymatic Activity and De-creased DNMT1Protein Levels.The next goal was to examine whether DOXO inhibits DNA methyltransferase en-zyme activity in the same concentration range in which it induces changes in reelin and GAD67mRNA levels.For this purpose,we used an in vitro enzymatic assay using nuclear extracts prepared from nontreated and DOXO-treated NT-2 cells.Extracts were used to measure methyltransferase ac-tivity with[3H]S-adenosylmethionine and an artificial DNA substrate.The data showed that100nM and250nM DOXO treatment of NT-2cells(48h)resulted in a significant70%and83%inhibition of nuclear DNMT activity,respectively (Fig.3).
To determine whether the reduction of DNMT enzymatic activity was due,at least in part,to decreased DNMT1pro-tein levels,we performed Western blot analyses.We observed a significant down-regulation of nuclear DNMT1protein af-ter100and250nM DOXO treatments(73%and83%,respec-tively,Fig.4,A and B).Although DNMT1protein is predom-inantly localized in the nucleus of NT-2cells,a small cytoplasmic fraction exists.The cytoplasmic DNMT1protein showed a similar trend toward decrease after DOXO treat-ment(data not shown).To assess whether DNMT1mRNA also showed a similar decrease,we examined the expression of DNMT1mRNA under the same conditions.The corre-sponding mRNA was not reduced by100and250nM DOXO (Fig.4,C and D),implying that DOXO-induced DNMT1 protein down-regulation is a post-transcriptional event.Be-cause it was of interest to determine whether the decrease in DNMT1protein levels paralleled temporally the induction of reelin and GAD67mRNAs,we performed time course West-ern blot analysis.As shown in Fig.4,E and F,100nM DOXO treatment led to a time-dependent decrease in DNMT1pro-tein levels.The reduction of DNMT1protein was apparent as early as6h after drug treatment(78%of the control
levels),
Fig.1.DOXO treatment leads to dose-
dependent increase in reelin and
GAD67mRNA levels.A,representa-
tive gels showing typical nonquantita-
tive RT-PCR analysis of reelin,
GAD67,and G3PDH mRNAs.B,bars
showing results of quantitative analy-
sis of reelin,GAD67,and G3PDH
mRNA levels in NT-2cells treated
with different concentrations of
DOXO for48h.Data are presented as
amount(picograms)of reelin,GAD67,
or G3PDH mRNA per1?g of total
RNA at the indicated concentrations
of DOXO(x-axis).C,dose-response
curves for reelin and GAD67mRNA
induction after48-h DOXO treatment
plotted as the log of drug concentra-
tion(x-axis).To compare reelin and
GAD67dose-response curves,the re-
sponse is expressed as a percentage of
the maximal reelin or GAD67mRNA
increase(y-axis).A baseline correction
was performed:y-axis values are nor-
malized so that the smallest(baseline)
mRNA values are defined as0%re-
sponse,whereas the highest values
(mRNA levels that correspond to max-
imal gene induction)are defined as
100%response.EC
50
is the effective
concentration of drug that leads to
50%of maximal reelin or GAD67gene
induction.D,representative gel show-
ing RT-PCR analysis of GAD65mRNA
expression in vehicle-or DOXO-
treated(250nM,48h)NT-2cells.
RNA isolated from untreated mouse
primary neuronal cultures and dis-
tilled water were used as positive(PC)
and negative controls(NC),respec-
tively.Data represent mean?S.E.M.
???,p?0.001;??,p?0.01versus
control group(one-way ANOVA fol-
lowed by Bonferroni test).
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whereas the amount of the protein decreased by half after 24h.
AZA and ZEB Treatments Induce Reelin and GAD67Gene Expression Associated with DNMT1Protein Depletion.To confirm that reelin and GAD67mRNAs are coordinately regulated through methylation-dependent mechanisms,we used two additional methyl-ation inhibitors,AZA and ZEB (Fig.5,A–D).NT-2cells were treated with 5?M AZA for 48h because it was shown previously that in the same cell system,this treatment leads to a significant increase in reelin mRNA levels (Chen et al.,2002),along with a decrease in reelin promoter methylation (Mitchell et al.,2005).As expected,AZA treat-ment increased reelin mRNA levels approximately 11-fold (from 0.0095?0.0018to 0.10?0.015pg of reelin mRNA per microgram of total RNA).However,in the current study,we showed that the same AZA treatment also led to a 10-fold induction of GAD67mRNA levels (from 0.12?0.011to 1.25?0.13pg of GAD67mRNA per microgram of total RNA).We also performed a dose-response study with ZEB,treating NT-2cells with the following concentrations
for 48h:0.5,5,50,and 500?M.As reported by another group using different cell lines,low concentrations of ZEB failed to induce their gene of interest (Cheng et al.,2004).Likewise,we found no changes in either reelin or GAD67mRNAs at low concentrations of ZEB (data not shown).However,at 500?M concentration of ZEB treatment (48h),the expression of reelin mRNA was increased approxi-mately 9-fold (0.09?0.004reelin mRNA per microgram of total RNA),whereas GAD67mRNA levels were elevated 2.5-fold (0.32?0.03pg of GAD67mRNA per microgram of total RNA).Because the previous study (Cheng et al.,2004)suggested that ZEB induced the demethylation of the p16gene with a 2-day delay,we also treated NT-2cells with 500?M ZEB for 48h and then allowed cells to grow in a fresh medium containing no drugs for an additional 48h before RNA isolation (2?2days).This treatment led to a more significant induction of both genes,with an almost 20-fold increase in reelin (0.19?0.02pg of reelin mRNA per microgram of total RNA)and 12.5-fold increase in GAD67mRNA levels (1.6?0.05pg of GAD67mRNA per microgram of total RNA).As in the case of DOXO treat-ment,neither AZA treatment nor ZEB treatment was as-sociated with significant changes in G3PDH mRNA levels (Fig.5B).However,all three treatments led to an almost complete depletion of DNMT1protein,as shown by West-ern blot analysis (Fig.5,C and D).
Activation of Reelin mRNA by DOXO Is Accompa-nied by the Dissociation of DNMT1and MeCP2and Increased Histone Acetylation from the Promoter Region.We next sought to understand the mechanisms by which DNMT inhibitors induce the reelin and GAD67genes.We examined changes at the level of the reelin promoter,because we had established previously cis -regu-latory elements that are operative in its regulation (Chen et al.,2002).We also have strong evidence showing that the reelin promoter in NT-2cells is silenced by methyl-ation,whereas the activation of the reelin gene corre-sponds with a decreased methylation of the https://www.sodocs.net/doc/97209073.html,-ing ChIP assays,we explored the possibility that inhibition of methylation results in a release of repressor proteins from the reelin promoter.Proteins and DNA were
first
Fig.2.DOXO treatment induces reelin and GAD67genes in a time-dependent manner.A,representative gels showing nonquantitative RT-PCR analysis of reelin,GAD67,and G3PDH mRNAs after DOXO treat-ment for the indicated times.B,bars showing the results of quantitative analysis using competitive RT-PCR and internal standards for the reelin,GAD67,and G3PDH mRNA levels in NT-2cells treated with 100nM DOXO for various times.Results are presented as amount (in picograms)of reelin,GAD67,or G3PDH mRNA per 1?g of total RNA.Data represent mean ?S.E.M.???,p ?0.001;?,p ?0.05versus control group (one-way ANOVA followed by Bonferroni
test).
Fig.3.DOXO reduces the DNA methyltransferase activity of NT-2cells.Total DNA methyltransferase activity of nuclear extracts from untreated or DOXO-treated calls was assayed in vitro by measuring the incorpora-tion of 3H-labeled methyl group donor S -adenosyl-L -methyl-methionine,into the DNA substrate [poly(dI-dC)?poly(dI-dC)oligonucleotide].Data are expressed as specific radioactivity (total radioactivity ?nonspecific radioactivity)normalized to the amount of the protein present in the corresponding nuclear extracts.Data represent mean ?S.E.M.???,p ?0.001versus control group (one-way ANOVA followed by Bonferroni test).
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cross-linked,and chromatin was sonicated to an average DNA fragment size from 200to 800bp (Fig.6A).Using specific antibodies for immunoprecipitation,we examined the association of MeCP2and DNMT1proteins with the promoter both before and after 48-h 250nM DOXO treat-ment.ChIP data showed that DNMT1and MeCP2are bound to the reelin promoter in untreated NT-2cells (Fig.6,B and C).In contrast,the induction of reelin mRNA by DOXO corresponded with a dissociation of these proteins from the promoter region (Fig.6,B and C).By using acetyl H3pull-down assays,we further explored whether changes in DNMT1and MeCP2binding were accompanied by changes in the acetylation status of histone H3in the vicinity of the promoter.As shown (Fig.6,B and C),DOXO significantly increased the amount of acetyl H3histone associated with the same region,implying that this treat-ment also alters chromatin structure in the vicinity of the reelin promoter.The specificity of these changes with the reelin promoter was demonstrated by amplifying the ?-glo-bin control region in parallel after pull-down assays using the same antibodies.As expected for a gene that is not epigenetically regulated,none of the examined proteins was bound to the ?-globin control region either before or after DOXO treatment (Fig.6B).Lack of DNMT1associa-tion with the reelin promoter correlated with almost com-plete depletion of DNMT1protein from NT-2cell nuclear extracts after 48-h 250nM DOXO treatment.However,neither 100nor 250nM 48-h DOXO treatment led to significant changes in MeCP2protein levels (Fig.6,D and E).
DOXO Treatments (100and 250nM for 48Hours)Are Not Associated with Significant NT-2Cell Loss.Besides acting as a DNMT1inhibitor,DOXO can act as a DNA-damaging agent that activates p53and induces apo-ptosis (Esteve et al.,2005).Previous studies using HCT116cells showed that only at a 1?10?6M concentration (and not lower),DOXO induced significant cell death that was related to apoptosis (Yokochi and Robertson,2004).Here we report that maximal induction of reelin and GAD67genes was associated with 250nM https://www.sodocs.net/doc/97209073.html,pared with EC 100treatment,the 1?M treatment was associated with slightly reduced reelin and GAD67mRNA levels (Fig.1B),which could be explained by the effect of DNA damage at that concentration of DOXO.To confirm that reelin and GAD67gene induction is not related to apoptosis,we per-formed cell viability assays after 100nM,250nM,and 2?M (48h)DOXO treatments.As shown in Fig.7,100and 250nM DOXO treatments were not associated with signif-icant NT-2cell death.However as anticipated,2?M DOXO induced considerable cell loss due to apoptosis and possibly to
necrosis.
Fig.4.DOXO down-regulates DNMT1protein levels post-transcriptionally.The representative Western immunoblots and the ratio of the DNMT1band over the area of the ?-actin band in nuclear extract protein samples from un-treated cells (control)and cells treated with ei-ther 100and 250nM DOXO for 48h (A and B)or 100nM DOXO for various times (E and F);1?corresponds to 2.5?g of protein;time course analysis was done using 5?g of protein.C,rep-resentative RT-PCR gels with internal stan-dards,and D,bars showing DNMT1mRNA lev-els in vehicle-treated and DOXO-treated cells,obtained using competitive RT-PCR assay.In each tube,competition was carried out among various concentrations of the DNMT1internal standard (IS)and 100ng of total RNA extracted from untreated cells (control)or cells treated with 100and 250nM DOXO for 48h.Data are presented as amount of DNMT1mRNA (in pi-cograms)per 1?g of total RNA.Data represent mean ?S.E.M.???,p ?0.001;?,p ?0.05versus control group (one-way ANOVA followed by Bonferroni test).
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Discussion
The data presented here clearly demonstrate that both
reelin and GAD67mRNA expression were significantly in-duced by three different DNMT inhibitors,namely DOXO, AZA,and ZEB.Most importantly,the detailed study with DOXO showed that this induction occurs1)in a similar dose-dependent manner(as shown by the same EC
50
and
EC
100
values for the induction of both mRNAs)and2)within the same time frame(both mRNAs begin to be induced after ?12h).The similar concentration-dependent and temporal activation patterns of the reelin and GAD67mRNAs strongly support our hypothesis that these two genes are coordinately regulated.Moreover,the finding that DOXO inhibits DNA methyltransferase activity in the same concentration range that induces reelin and GAD67mRNA expression provides additional evidence that reelin and GAD67genes are acti-vated epigenetically.However,the assay we used measures total DNA methyltransferase activity and probably reflects the activities of so-called maintenance methyltransferase (DNMT1)and the activity of de novo methyltransferases (DNMT3A and DNMT3B).Using recombinant DNMT1pro-tein,Yokochi and Robertson(2004)previously showed that DOXO inhibits the enzymatic activity of DNMT1.As indirect evidence that this drug inhibits DNMT1enzymatic activity under the conditions that we applied,we showed that the reduction of total DNMT activity is highly correlated with the reduction in DNMT1protein levels following the same DOXO treatment.Furthermore,AZA and ZEB treatments,which induced reelin and GAD67mRNAs,led to a complete deple-tion of nuclear DNMT1protein.Consistent with this,a pre-vious study of ours demonstrated that the knockdown of DNMT1protein is associated with an up-regulation of mouse reelin and GAD67mRNA levels in cortical neurons in vitro (Noh et al.,2005).Taken together,these data imply a possi-ble role for DNMT1in the coordinated regulation of the reelin and GAD67genes.Having said this,we cannot exclude the possibility that DNMT3A and/or3B might also play a role in these events.
In dividing cells such as NT-2cells,DNMT1is believed to be mainly involved in the methylation of hemimethylated DNA.This process predominates during DNA replication. Therefore,drugs that inhibit DNMT1enzymatic activity, such as DOXO,AZA,and ZEB,may require several cell divisions to induce significant changes in promoter methyl-ation status,along with changes in mRNA expression(Egger et al.,2004).It is interesting that we observed an induction
of
Fig.5.The effects of AZA and ZEB
treatments on reelin and GAD67
mRNA expression and DNMT1pro-
tein levels.A,representative gels of
nonquantitative RT-PCR analysis
and B,bars showing the results of
quantitative analysis of the reelin,
GAD67,and G3PDH mRNA levels in
NT-2cells treated with5?M AZA for
48h and500?M ZEB for either48h
(ZEB48h)or48h followed by48-h
incubation with untreated medium
(ZEB2?2D).Results are presented
as the amount(in picograms)of ree-
lin,GAD67,or G3PDH mRNA per mi-
crogram of total RNA.C,representa-
tive DNMT1and?-actin Western
immunoblots,and D,the ratio of the
DNMT1band over the area of the
?-actin band in nuclear extract pro-
tein samples of control and AZA-or
ZEB-treated NT-2cells(1?corre-
sponds to5?g of protein).Data rep-
resent mean?S.E.M.???,p?0.001;
??,p?0.01versus control group(one-
way ANOVA followed by Bonferroni
test).
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both reelin and GAD67mRNAs as early as12h after initi-ating DOXO treatment.In contrast,20to24h are needed to complete one cell cycle.This observation led us to consider a possible corepressor role of DNMT1protein in regulating reelin and GAD67mRNA expression.It has been shown that DNMT1represses gene transcription through its noncata-lytic domain independent of its methyltransferase function. This action occurs through the recruitment of MeCP2and HDACs(Fuks et al.,2000;Burgers et al.,2002;Kimura and Shiota,2003).As an example,it has been shown recently that DNMT1can suppress the activity of the metallothionein-I gene promoter regardless of its methylation status(Majum-der et al.,2006).To explore whether this is also the case with the reelin and GAD67genes,we first checked the time frame of DNMT1protein down-regulation.It is striking that DNMT1protein levels start to decrease6h before the induc-tion of reelin and GAD67mRNAs occurs.Our data demon-strated that the down-regulation of DNMT1occurs post-transcriptionally,because we show that DOXO does not induce changes in DNMT1mRNA levels.It seems likely that DNMT1may get trapped in a DOXO-DNA complex,which subsequently targets DNMT1for degradation(Yokochi and Robertson,2004).This event can be replication-independent, because it has been shown that in addition to S phase, DNMT1is continuously loaded onto chromatin during the G
2 and M phases of the cell cycle(Easwaran et al.,2004).In support of our finding,another group reported that2h of AZA treatment is sufficient to induce significant replication-independent reduction in DNMT1protein levels(Ghoshal et al.,2005).Furthermore,we demonstrated that DNMT1pro-tein decreases in a time-dependent fashion,very similar to that seen for the increases in reelin and GAD67mRNA levels.
As additional evidence for the corepressor role of DNMT1, we showed(using ChIP assays)that this protein is bound to the reelin promoter when the gene is silent or transcription-ally inactive.In contrast,the maximal induction of the reelin mRNA is accompanied by a complete dissociation of DNMT1 from the promoter regulatory region.These data suggest that DNMT1is involved in keeping the reelin promoter in a re-pressed state in NT-2cells.It seems likely that the slight activation of the reelin(and probably GAD67)gene(s),seen 12h after beginning DOXO treatment,is triggered by de-creased amounts of DNMT1in these cells and the subsequent decreased binding of DNMT1to the reelin promoter.This,in turn,might lead to the release of the repressor complex from the reelin promoter.Evidence suggests that this repressor complex probably includes MeCP2and certain HDACs.It has been shown recently that the down-regulation of reelin and GAD67mRNAs corresponds with increased recruitment of MeCP2to the mouse reelin and GAD67promoters(Dong et al.,2005).Here we demonstrate that the maximal
activation
Fig.6.DOXO induces dissociation of DNMT1and MeCP2and increased acetylation of H3histones in the reelin promoter region.A,chromatin samples containing200-to800-bp DNA fragments were generated from cells treated with either control medium(C,control)or250nM DOXO for48h(D, DOXO).B,for both treatments,the reelin promoter region(482-bp band)and?-globin gene fragments(289-bp band)were PCR-amplified from nonimmunoprecipitated input(1:10and1:4dilutions),samples immunoprecipitated with DNMT1antibody(DNMT1IP),MeCP2antibody(MeCP2IP)
or anti-acetyl histone H3antibody(Ac-H3IP),and negative control(no antibody).C,results of semiquantitative analysis of the occupancy of DNMT1
and MeCP2to the reelin promoter in vehicle-and DOXO-treated cells normalized to input DNA(1:4dilution).For comparison,the amounts of acetylated histone H3is shown after treatment.Data are presented as a ratio of relative optical densities(ROD)of the bands within the immunoprecipitated sample(IP)and input lanes derived from ethidium bromide-stained gels.Data represent mean?S.E.M.???,p?0.001.Control versus DOXO(Student’s t test).D,representative MeCP2and?-actin Western immunoblots,and E,the ratio of the MeCP2band over the area of the
?-actin band from nuclear extract protein samples of vehicle-treated(control)and DOXO-treated NT-2cells(1?corresponds to5?g of protein).Data represent mean?S.E.M.(one-way ANOVA followed by Bonferroni test).
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of the reelin gene by DOXO is associated with the dissocia-tion of MeCP2from the promoter region and an increase in H3histone acetylation in the vicinity of the promoter.Be-cause MeCP2binds specifically to methylated cytosines,our data also suggest that 48h of DOXO treatment induces changes in the methylation status of the reelin promoter.This conclusion is strengthened by the finding that unlike DNMT1,MeCP2protein levels did not change after the same treatment.We have shown previously that all treatments that induce reelin expression,including AZA,also decrease reelin promoter methylation (Chen et al.,2002;Mitchell et al.,2005).However,as noted previously,12-h treatment that produces a slight induction of reelin and GAD67mRNAs seems likely to be insufficient to induce changes in promoter methylation status.Therefore,we suggest that promoter methylation per se may not be sufficient to keep the reelin promoter in a fully repressed https://www.sodocs.net/doc/97209073.html,plete silencing of the reelin promoter probably requires the fully assembled re-pressor complex and highly condensed chromatin maintained by the recruitment of DNMT1.However,we believe that the reelin promoter must be demethylated for the maximal acti-vation of the gene to occur.Additional studies are needed to confirm this speculation.
In conclusion,we highlight several implications of these data.First,the study suggests a mechanism by which reelin and GAD67mRNAs might be coordinately regulated in GABAergic neurons of the adult brain.It seems likely that both genes may be regulated by methylation of the corre-sponding promoters.DNMT1probably has a dual role in this process.One could be its well-established enzymatic (DNA methyltransferase)role,by which it controls the methylation status and the activity level of the reelin,GAD67,and pos-sibly other epigenetically regulated promoters.Another role of DNMT1could be to participate in the formation of the transcriptional repressor complex by recruiting MeCP2,HDACs,and other corepressors.This may lead to the gener-ation of a more condensed chromatin structure that subse-quently limits promoter accessibility.As mentioned above,although we have focused on the role of DNMT1in this process,we cannot exclude a contributing role for either DNMT 3A and or DNMT 3B.
Second,this study gives new insight into the molecular mechanisms that underlie the down-regulation of reelin and GAD67mRNAs in the brains of patients with schizophrenia.We propose that the reported up-regulation of DNMT1(Vel-dic et al.,2004)leads to the hypermethylation and increased binding of DNMT1to the reelin and GAD67promoters.Fur-thermore,we suggest that there is a subsequent increased recruitment of MeCP2,HDACs,and possibly additional core-pressor proteins.However,studies with postmortem human brains will be necessary to confirm this hypothesis.Third,we suggest a new approach in the treatment of schizophrenia that focuses on the reactivation of expression of genes that are down-regulated due to modifications in the epigenome.Thus far,epigenetic drugs (DNA methylation inhibitors and HDAC inhibitors)have been used in cancer treatment,be-cause they often selectively reactivate tumor suppressor genes that are silenced by CpG island promoter methylation (Egger et al.,2004).It seems likely that this may be one of the mechanisms that contributes to the therapeutic benefits of DOXO in some types of cancer.However,of specific interest in the context of schizophrenia research,we report that DOXO concentrations that do not induce significant cell death lead to a robust induction of the reelin and GAD67mRNAs.Furthermore,our data suggest that DOXO induces changes in the methylation status of the reelin promoter,which has been shown to be hypermethylated in the brains of patients with schizophrenia (Abdolmaleky et al.,2005;Gray-son et al.,2005).Although this remains to be addressed experimentally,the changes in methylation most likely occur only at specific promoters,because another group reported no changes in global methylation of genomic DNA after DOXO treatment (Yokochi and Robertson,2004).We propose that drugs that induce promoter hypomethylation and/or DNMT1down-regulation might be useful in correcting the reelin and GAD67mRNA insufficiencies associated with schizophrenia.This means that DNMT1and HDACs may represent possible new molecular targets to treat patients with schizophrenia.At the same time,because many of these drugs are toxic to cells and may have global effects in the nervous system that have yet to be determined,the safety of these compounds needs to be fully tested in animal models before adopting their use in humans.
Acknowledgments
We dedicate this article to the memory of our close friend and scientific colleague Robert H.Costa,who died September 1,2006,from complications associated with pancreatic cancer.
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Address correspondence to:Dr.Dennis R.Grayson,Department of Psychi-atry,College of Medicine,University of Illinois at Chicago,1601W.Taylor St.,Chicago IL,60612.E-mail:dgrayson@https://www.sodocs.net/doc/97209073.html,
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DNA甲基化实验操作原理及方法-Hxg
DNA 甲基化重亚硫酸氢盐修饰法（DNA METHYLATION BISULFITE MODIFICATION） 实验操作原理及方法 一、实验目的： 通过本实验，可以检测特定DNA序列的甲基化状态。 二、实验原理： DNA 甲基化是指由S-腺苷甲硫氨酸（SAM）提供甲基基团，在DNA 甲基转移酶（DNA methyltransferases，DNMTs）的作用下，将CpG 二核苷酸的胞嘧啶（C）甲基化为5-甲基化胞嘧啶（5-m C）的一种化学反应。DNA 甲基化是调节基因转录表达的一种重要的表观遗传的修饰方式。 DNA 甲基化主要在转录水平抑制基因的表达。DNA 甲基化引起基因转录抑制的机制可能主要有以下3 种：（1）DNA甲基化直接干扰特异性转录因子与各基因启动子中识别位置的结合。（2）序列特异性的甲基化DNA 结合蛋白与启动子区甲基化CpG 岛结合，募集一些蛋白，形成转录抑制复合物，阻止转录因子与启动子区靶序列的结合，从而影响基因的转录。（3）DNA 甲基化通过改变染色质结构，抑制基因表达。 重亚硫酸氢盐修饰法检测DNA甲基化的基本原理是基于DNA变性后用重亚硫酸氢盐处理，可将未甲基化胞嘧啶修饰成尿嘧啶。此反应的步骤是：1、在C-6位点磺化胞嘧啶残基；2、在C-4处水解去氨基来产生尿嘧啶磺酸盐；3、在碱性条件下去硫酸化。在这个过程中，5-甲基胞嘧啶由于甲基化基团干扰了重亚硫酸氢盐进入到C-6位点而保持着未反应的状态。在重亚硫酸氢盐处理后，使用针对每个修饰后DNA链的引物进行PCR反应。在这个PCR产物中，每5-甲基胞嘧啶显示为胞嘧啶，而由未甲基化胞嘧啶转变成的尿嘧啶则在扩增过程中被胸腺嘧啶所取代。 BSP(bisulfate sequencing PCR) ：重亚硫酸盐使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶，而甲基化的胞嘧啶保持不变，进行PCR扩增。最后，对PCR产物进行测序，并且与未经处理的序列比较，判断是否CpG位点发生甲基化。
免疫组化步骤
免疫组化实验步骤 细胞和组织的固定 （一）固定 为了更好的保持细胞和组织原有的形态结构，防止组织自溶，有必要对细胞和组织进行固定。固定的作用不仅是使细胞内蛋白质凝固，终止或抑制外源性和内源性酶活性，更重要的是最大限度的保存细胞和组织的抗原性，使水溶性抗原转变为非水溶性抗原，防止抗原弥散。不同抗原，其稳定性也不相同，因而对固定剂的耐受性差异较大 （二）固定剂 用于免疫组织化学的固定剂种类较多，性能各异，在固定半稳定性抗原时，尤其重视固定剂的选择，介绍如下。 1．醛类固定剂双功能交联剂，其作用是使组织之间相互交联，保存抗原于原位，其特点是对组织穿透性强，收缩性小。有人认为它对IgM、IgA、J链、K 链和λ链的标记效果良好，背景清晰，是常用的固定剂。 1）4%多聚甲醛为我们常用固定剂 2）Bouin’s液 该固定液为组织学、病理学常用固定剂之一，对组织穿透力较强而收缩性较小，比单独醛类固定更适合免疫组化染色。Kayhko认为用于标记B细胞的J链较好，但Bullock则认为它可导致Igg γ重链变性，故必需加大第一抗体的浓度。 2．丙酮及醇类固定剂 系最初免疫细胞化学染色的固定剂，其作用是沉淀蛋白质和糖，对组织穿透性很强，保存抗原的免疫活性较好。但醇类对低分子蛋白质、多肽及胞浆内蛋白质保存效果较差，解决的办法是和其它试剂混合使用，如加冰醋酸、乙醚、氯仿、甲醛等。 丙酮的组织穿透性和脱水性更强，常用于冰冻切片及细胞涂片的后固定，保存抗原性较好，平时4。C低温保存备用，临用时，只需将涂片或冰冻切片插入冷丙酮内5-10min，取出后自然干燥，贮存于低温冰箱备用。 以上介绍了免疫组织化学中常用的固定液，用于免疫组化的固定剂种类很多，不同的抗原和标本需经过反复试验，选用最佳固定液，迄今尚无一种标准固定液可以用于各种不同的抗原固定。而且同一固定液固定的组织，免疫组化染色标记
甲基化检测原理及步骤 DNA实验技术方法汇总
DNA亚硫酸氢盐修饰和纯化操作步骤修饰设计：使用CpGenome TM kit使胞嘧啶转化为尿嘧啶的步骤如下。中等温度碱性pH下使DNA变性成为单链形式暴露出碱基。试剂一，一种包含亚硫酸氢根的钠盐，可使未甲基化的胞嘧啶磺化和水解脱氨，产生一种尿嘧啶磺酸盐中间产物。然后DNA在另一种盐﹙试剂二﹚存在的条件下与一种微粒载体﹙试剂三﹚结合，并通过重复离心和在70%的乙醇中重悬浮脱盐。向尿嘧啶的转化是通过在90%的乙醇中反复碱性脱磺酸基作用和脱盐完成的。DNA最终在TE缓冲液中通过加热从载体上洗脱下来。 第一步：试剂准备 （1）3 M NaOH原料（用前现配） 把1g干NaOH片剂溶解在8.3mL水中。使用此类腐蚀性碱，注意小心谨慎和实验操作。 （2）20 mM NaOH/90% EtOH（用前现配） 配制1mL该溶液需：900μl 100%的乙醇，93.4μl水，6.6μl 3M的氢氧化钠。 （3）溶解试剂Ⅰ（用前现配） 打开前将试剂瓶加温至室温。对每份待修饰的样本，称取0.227g DNA修饰试剂Ⅰ加入0.571mL水中。充分涡旋振荡混合。使用该试剂时要小心谨慎，因为它对呼吸系统和皮肤有刺激性。用大约20μl 3M NaOH调整pH至5.0，用pH试纸检测pH值。试剂Ⅰ避光保存以免分解。为了最佳效果，试剂应在配置后立即使用。 （4）溶解试剂Ⅱ 打开前将试剂瓶加温至室温。将1μl β-巯基乙醇加入20mL去离子水中。每份待修饰的DNA样本需将750μl该溶液加入到1.35g DNA修饰Ⅱ。充分混合确保完全溶解。过量的试剂可用箔纸包裹的容器、2℃-8℃、避光保存长达6周。 第二步：DNA修饰程序 1、在带有螺旋形瓶盖的1.5-2.0mL的微量离心管中：将7.0μl 3M NaOH加入到含有1.0 μg DNA的100μl水中（10ng/μl），混匀。 注意：如果样本含有的DNA量不到1.0μg，就向样本DNA中加入2 μl DNA修饰试剂Ⅳ并加水至总体积100μl。再加入7.0μl 3M NaOH并混匀。 2、50℃ DNA孵育10分钟（加热块或水浴）
DNA甲基化——试剂盒+抗体解决方案
DNA甲基化——试剂盒+抗体解决方案 DNA甲基化（DNA methylation）为DNA化学修饰的一种形式，能够在不改变DNA序列的前提下，改变遗传表现。所谓DNA甲基化是指在DNA甲基化转移酶的作用下，在基因组CpG二核苷酸的胞嘧啶5号碳位共价键结合一个甲基基团。大量研究表明，DNA甲基化能引起染色质结构、DNA构象、DNA稳定性及DNA与蛋白质相互作用方式的改变，从而控制基因表达。 一、DNA甲基化修饰相关产品 DNA甲基化修饰研究手段——DNA亚硫酸盐转化，使用亚硫酸氢钠将胞嘧啶转化为尿嘧啶，而5-甲基胞嘧啶（5-mC）保持完整。即未甲基化的胞嘧啶残基被脱氨成尿嘧啶，甲基化的胞嘧啶（5-mC）残基不受影响，这使PCR扩增可将尿嘧啶视为胸腺嘧啶，将5-mC或5-hmC识别为胞嘧啶。这样便能够区分甲基化和未甲基化的胞嘧啶残基，从而提供有关DNA甲基化区域的单核苷酸分辨率信息。 要成功地进行DNA甲基化研究，必须进行完全转化，并减少通常由于严酷的化学反应而导致的DNA降解量。基于亚硫酸氢盐和亚硫酸氢钠的方法是用于研究DNA甲基化并帮助制备基因组DNA进行基因特异性DNA甲基化分析的常用方法。亚硫酸氢盐转化后通常是下游应用，在基因特异性基础上分析DNA甲基化的流行下游方法包括亚硫酸氢盐测序，甲基化特异性PCR（MS-PCR）和基于甲基化的微阵列。 整个转换过程中，高质量的DNA是至关重要的，因为转换过程中的酸性物质会破坏DNA。而对于大规模亚硝酸氢盐转化实验，高通量选择对于节省时间和降低成本至关重要。 此外，还有高通量DNA修饰试剂盒，EpiNext高灵敏度亚硫酸氢盐测序试剂盒（Illumina），一步法DNA 修饰试剂盒，Methylamp通用甲基化DNA试剂盒，Methylamp全细胞亚硫酸氢盐修饰试剂盒等。
DNA甲基化分析方法
· 489 · 《生命的化学》２００８年２８卷４期ＣＨＥＭＩＳＴＲＹ ＯＦ ＬＩＦＥ ２００８，２８（４） ● 技术与方法 文章编号： １０００－１３３６（２００８）04－0489-03 ＤＮＡ甲基化分析方法 肖正中 邬苏晓 （韶关学院英东生物工程学院，韶关 ５１２００５） 摘要：ＤＮＡ甲基化是表观遗传学的重要研究内容之一。甲基化分析的方法多且研究难度大，各种方法都有其一定的优势和不足。本文综述了基因组ＤＮＡ甲基化和特定ＤＮＡ片段甲基化状态分析方法新进展，为研究者提供参考。关键词：ＤＮＡ甲基化；ＣｐＧ岛；表观遗传学中图分类号：Ｑ３３４ 收稿日期：２００８－０４－１２ 作者简介：肖正中（１９７２－），男，博士，讲师，Ｅ－ｍａｉｌ：ｘｚｚｗｓｘ＠ｓｉｎａ．ｃｏｍ；邬苏晓（１９７２－），女，硕士，讲师，联系作者，Ｅ－ｍａｉｌ：ｓｘｗａ４０３５＠ｓｉｎａ．ｃｏｍ ＤＮＡ甲基化是表观遗传学的重要研究内容之一，它可以在转录水平抑制基因的表达。甲基化通常发生在胞嘧啶的Ｃ５位，形成５－甲基胞嘧啶（５ｍＣ），甲基化的胞嘧啶多位于ＣｐＧ岛上。ＣｐＧ岛是ＣｐＧ二联核苷富集区域，ＣＧ含量大于５０％，长约２００￣５００ ｂｐ。哺乳动物ＤＮＡ甲基化的模式只有５ｍＣ这一形式，真核生物中大约２％￣７％的胞嘧啶被甲基化修饰。１． 基因组ＤＮＡ甲基化分析方法 早期的基因组ＤＮＡ甲基化分析技术，如ＳｓｓＩ甲基转移酶分析法、氯乙醛反应法、免疫学抗体技术等，已不能满足现代表观遗传学研究的需求。近年来常用的基因组甲基化分析方法有以下两种。１．１ 甲基化敏感扩增多态性技术 甲基化敏感扩增多态性（ｍｅｔｈｙｌａｔｉｏｎ ｓｅｎｓｉｔｉｖｅ ａｍｐｌｉｆｉｃａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍ，ＭＳＡＰ）技术由Ｒｅｙｎａ－ｌóｐｅｚ等报道，并被用于检测双相型真菌的ＤＮＡ甲基化［１］ ，它 是在扩增片段长度多 态性（ａｍｐｌｉｆｉｅｄ ｆｒａｇｍｅｎｔ ｌｅｎｇｔｈ ｐｏｌｙｍｏｒｐｈｉｓｍ， ＡＦＬＰ）技术的基础上建立起来的。其基本程序是：提取高质量基因组ＤＮＡ，分别用ＥｃｏＲＩ／ＨｐａＩＩ，ＥｃｏＲＩ／ＭｓｐＩ两组酶组合对基因组ＤＮＡ进行双酶切，并连上相应的限制性内切酶的接头，然后以接头序列设计的预扩增引物，进行ＰＣＲ扩增。扩增产物稀释后，再加入带有选择性碱基的引物，进行第二次ＰＣＲ扩增，扩 增产物变性后在６％的序列胶上电泳，最后采用银染或同位素放射自显影方法处理序列胶，统计和分析ＤＮＡ条带。这种方法在研究动植物基因组甲基化上有广泛应用［２－６］。 ＭＳＡＰ技术相对其他测定ＤＮＡ甲基化程度的技术有如下优点：（１）不需知道被测ＤＮＡ的序列信息，在不同生物上具有通用性，可用于ＤＮＡ序列背景知识未知的生物。（２）操作相对简便，在ＡＦＬＰ技术体系的基础无需改进，即可操作。（３）可在全基因组范围检测ＣＣＧＧ位点的胞嘧啶甲基化变化。ＭＳＡＰ技术的局限性在于不能完成非ＣＣＧＧ位点的胞嘧啶甲基化。１．２ 高效液相层析及相关方法 高效液相层析（ｈｉｇｈｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ， ＨＰＬＣ）能够定量测定基因组整体甲基化水平，其过程是：先将ＤＮＡ样品经盐酸或氢氟酸水解成碱基，水解产物通过色谱柱，将结果与标准品比较，紫外光测定吸收峰值，计算５ｍＣ／（５ｍＣ＋５Ｃ）的积分面积得出基因组整体的甲基化水平。Ｆｒａｇａ等［７］运用高效毛细管电泳法（ｈｉｇｈ ｐｅｒ－ｆｏｒｍａｎｃｅ ｃａｐｉｌｌａｒｙ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ， ＨＰＣＥ）处理ＤＮＡ水解产物确定５ｍＣ的水平，相比ＨＰＬＣ，ＨＰＣＥ更简便、快速、经济。ＨＰＬＣ及ＨＰＣＥ测定基因组整体ＤＮＡ甲基化水平的敏感性均较高。２． 特定ＤＮＡ片段甲基化检测方法 ２．１ 亚硫酸氢盐预处理法 （１）甲基化特异性ＰＣＲ。甲基化特异性ＰＣＲ（ＭＳＰ）是Ｈｅｒｍａｎ等［８］首先提出的一种 检测基因组ＤＮＡ甲基化水平的常用方法。该法是将ＤＮＡ经亚硫酸氢钠处理，非甲基化的胞嘧啶转变为尿嘧啶，而甲基化的胞嘧啶保持不变。在ＰＣＲ反应
免疫组化超详细步骤
免疫组化超详细步骤 Prepared on 22 November 2020
免疫组化 一实验目的：分别用KRAS、NRAS、HRAS蛋白抗体检测肺癌组织中KRAS、NRAS、HRAS蛋白的表达情况 二实验原理:应用基本原理——抗体反应，即抗原与抗体特异性结合的原理，通过化学反应使的（、酶、、）显色来确定组织细胞内抗原（多肽和），对其进行定位、定性及定量的研究，称为或. 三实验试剂及耗材:经病理医生确认的肺癌组织切片、KRAS、NRAS、HRAS蛋白抗体、DAB显色试剂盒、抗原修复液、PBS、苏木素染料、1%盐酸酒精溶液、%氨水、二甲苯、等 四实验设备：切片机、4℃冰箱、显微镜、烤片机 五主要试剂配置: 缓冲液 应用液配成1000毫升溶液 应用液BNa2HPO4·配成1000毫升溶液 配制1000毫升PBS需要14毫升应用A液，36毫升应用B液，加。 枸橼酸钠抗原修复液 应用液A枸橼酸配成1000毫升溶液 应用液B柠檬酸三钠（三水合柠檬酸三钠）配成1000毫升溶液 配制500毫升抗原修复液需要9毫升应用A液，41毫升应用B液。 苏木素染液配方：苏木素2g,无水乙醇250毫升，硫酸铝蒸馏水750毫升，碘酸钠，冰醋酸20毫升。先将苏木素溶于无水乙醇，再将硫酸铝溶于蒸馏水水中。两液溶解后将其混合，加入碘酸钠，最后加入冰醋酸。 抗体说明书建议的抗体稀释度 N-Ras1:50-1:500 H-Ras1:50-1:500 K-Ras1:20-1:200 六实验步骤： 1组织常规石蜡切片厚度3-5um，防脱片捞片后晾干，放入72℃烤箱烤片2小时。 2切片脱蜡水化程序 ⑴二甲苯Ⅰ、Ⅱ脱蜡各12分钟； ⑵无水乙醇Ⅰ、Ⅱ各3分钟；（洗二甲苯） ⑶95%乙醇3分钟； ⑷85%乙醇3分钟； 3自来水漂洗3分钟，洗涤一定要充分。 4组织修复采用高温高压法：压力锅中加入，抗原修复液约1000ml，切片插入塑料架上，放入压力锅，盖上锅盖（此时不扣上压力阀）1600W预热至沸腾，扣上压力阀1300W，待高压锅阀门喷气开始计时，修复时间为2分钟。 5停止加热并用流水冲洗高压锅以降温，室温冷却。 6将抗原修复后切片置自来水中，浸泡2分钟。将样本置于内源性过氧化物酶阻断剂 3%H2O2，室温放置4分钟。（需要盖盖子）自来水洗2分钟，PBS缓冲液洗涤2分钟。
甲基化原理及方法
甲基化检测方法（亚硫酸氢盐修饰测序法） 基本原理在于：样本经亚硫氢酸盐处理后,甲基化的胞嘧啶（C）保持不变,但非甲基化的胞嘧啶被转化成脲嘧啶,因此在利用该处理产物作为模板的PCR产物中,甲基化的胞嘧啶还是胞嘧啶,但非甲基化胞嘧啶变成了脲嘧啶（胸腺嘧啶）,此时检测到的胞嘧啶（C）即是样品中本身的甲基化位点. 第一部分基因组DNA的提取。 DNA提取试剂盒，如果实验室条件成熟，自己配试剂提取完全可以。DNA比较稳定，只要在操作中不要使用暴力，提出的基因组DNA应该是完整的。 此步重点在于DNA的纯度，即减少或避免RNA、蛋白的污染很重要。因此在提取过程中需使用蛋白酶K及RNA酶以去除两者。 使用两者的细节： 1：蛋白酶K可以使用灭菌双蒸水配制成20mg/ml； 2：RNA酶必须要配制成不含DNA酶的RNA酶，即在购买市售RNA酶后进行再处理，配制成10mg/ml。否则可能的后果是不仅没有RNA，连DNA也被消化了。两者均于-20度保存。 验证提取DNA的纯度的方法有二： 1：紫外分光光度计计算OD比值； 2：1%-1.5%的琼脂糖凝胶电泳。 我倾向于第二种方法，这种方法完全可以明确所提基因组DNA的纯度，并根据Marker的上样量估计其浓度，以用于下一步的修饰。 第二部分亚硫酸氢钠修饰基因组DNA 如不特别指出，所用双蒸水（DDW）均经高压蒸汽灭菌。 1：将约2ugDNA于1.5mlEP管中使用DDW稀释至50ul； 2：加5.5ul新鲜配制的3M NaOH； 3：42℃水浴30min； 水浴期间配制： 4：10mM对苯二酚（氢醌），加30ul至上述水浴后混合液中；（溶液变成淡黄色） 5：3.6M亚硫酸氢钠（Sigma，S9000），配制方法：1.88g亚硫酸氢钠使用DDW稀释，并以3M NaOH滴定溶液至PH 5.0，最终体积为5ml。这么大浓度的亚硫酸氢钠很难溶，但加入NaOH后会慢慢溶解，需要有耐心。PH一定要准确为5.0。加520ul至上述水浴后溶液中。 6：EP管外裹以铝箔纸，避光，轻柔颠倒混匀溶液。 7：加200 ul 石蜡油，防止水分蒸发，限制氧化。 8：50℃避光水浴16h。 一般此步在4pm开始做，熟练的话不到5pm即可完成，水浴16h正好至次日8am以后收，时间上很合适。 这一步细节： 1：基因组DNA的量不需十分精确，宁多勿少，因为在以后纯化回收步骤中会有丢失，且此方法修饰最多可至4ug。
甲基化--经验
Ensembl data bank 甲基化测序BSP法的那个黑白点状图（黑表示甲基化、白点表示非甲基化）是怎么做出来的呀上 用BiQ ANALYZER软件，免费的可以下载，安装需要最新的java程序 ，关于后续的一些步骤，我看了一篇国内的硕士论文，如下： 2.2.1.4.6连接反应产物的转化 (l)每个连接反应准备1个含有氨节青霉素的LB平板，涂板前半小时将平板从冰箱 中取出平衡至室温。 (2)离心使连接反应内容物汇集到管底，吸取10ul连接反应产物加到置于冰上的 1.5ml离心管中. (3)将冻存的JM109高效率感受态细胞从一70℃冰箱中取出，放置在冰浴直至融化 (大概5分钟)，轻轻振动离心管使之混匀。 (4)向步骤2准备的每个转化管中加入50ul感受态细胞。 (5)轻轻振动小管混匀，冰浴30分钟。 (6)在精确的42℃水浴中热击45一50秒(不要振动)。 (7)迅速将管子移到冰浴中，使细胞冷却2分钟。 (8)每管连接反应转化细胞中加入平衡至室温的200ulLB培养基， (9)在37℃振荡培养(150rpm)1小时。 (10)将每个转化培养基200ul涂到LB/氨苄/IPTG/X-Gal平板上。 (11)将平板于37℃恒温箱中过夜培养(16一24小时)。 2.2.L4.7阳性克隆筛选 从培养箱中取出平板，置于4℃冰箱使蓝色充分显现。挑取白斑菌落到5mLB培 基，37℃摇床上培养过夜。吸取lml菌液送测序，引物为通用引物M13。 进入丁香园，是从做MSP开始的。从对DNA甲基化的一无所知到MSP实验成功，经历了很多艰难，同时也收获得了不少经验。从丁香园的帖子可以看出，近几年来，国内对DNA 甲基化的研究在逐年增加，战友们在实验中遇到的困难也不少。在这里谈一下对MSP,及BSP的一点体会，希望能对新手们有所帮助，也请老手们批评补充。 亚硫酸氢盐转化和PCR扩增是MSP,BSP的两个基本步骤。亚硫酸氢盐转化，估计现在做手工修饰的也不多了，试剂盒能够很方便的帮我们解决问题。但PCR中的引物设计，现在可能还是个难题。看到很多帖子里提到的引物都是来源于文献的，但事实上在很多情况下，我们是没法找到现存引物的，因此掌握BSP,MSP引物的设计就显得非常必要了。 BSP，MSP引物设计的一个最大问题在于亚硫酸氢盐的转化使得模板的序列复杂性大大降低，而且经常产生T或G或者富含GC片段交替出现的序列。这个会使引物选择变得困难。因此，往往我们需要设计巢式引物来解决这个问题。BSP,MSP引物的设计，除了要遵循引物设计的基本原则外，还要注意一下一些规则：
免疫组化操作方法原理步骤以及常见问题处理大总结
免疫组化操作方法、原理、步骤以及常见问题处理大总结 1、方法操作不难，最大的难处是出现异常结果时如何解决？这就需要掌握免疫组化实验原理，每一步知道为什么这样做，这样你才敢大胆地改革先前的不对的方法步骤。如抗体孵育条件主要是抗体浓度、温度、时间，这三者一般是相互成反比的（相对），其中浓度是最重要的先决条件，温度决定反应的速度、时间决定反应的量。就拿温度来说，可以有4度、室温、37度，我推荐4度最佳，反应最温和，背景较浅；而37度反应速度较快，时间较短；室温我不太提倡，除非你每次都把环境温度控制在一定的范围，否则，尽量选择前两者。 2、免疫组化最大的优势是定位和定性。相比于其他蛋白检测方法，免疫组化具有定性灵敏度高、定位较直接准确，是定位检测分析首选方法。尤其对于有些因子的转位研究十分有用。 3、免疫组化结果定量分析的前提是高质量的染色切片。免疫组化结果也能定量分析，但必须是背景染色浅而特异性染色较深的情况下，分析最为准确，这种原则可能也是我们日常审稿时判定研究结果的必备条件。 4、免疫组化实验一定要设置阳性对照和阴性对照。阳性对照一般是用肯定表达这种抗原的切片来做；阴性对照一般是用PBS或非一抗替代一抗来进行反应，其余步骤均一致。前者是排除方法和实验系统有无问题；后者是排除有无一抗外的非特异性染色。 5、免疫组化的应用广泛，是当前实验研究的最重要方法之一。如今发SCI论文时，明显感觉仅靠量化的数据来发文章很难，加一些形态学数据或图片，老外十分欢迎，可能是怕你学术造假吧。当然也不能做假阳性或假阴性结果。 6、免疫组化技术掌握与否的鉴定标准是同一切片或不同切片中不同抗原均从摸索浓度或条件而做出优良的染色切片。我在平时带教中就发现许多研究生把我已经摸索很成熟的反应条件、浓度、方法步骤，重复运用于同一性质的切片和同一种抗体，做出来后就觉得自己已经掌握了免疫组化方法，更换一种抗体后，居然连二抗的种属来源都拿错了。失败往往促进你去思考试验原理和过程，成功有时也加快你自傲。 7、实验方法需要动手+动脑。如今我还不敢说我在免疫组化什么都知道。我只所以今天敢在这里说这说那，这是因为我经过了反复的动手+动脑，把理论原理运用于实践，在把实践中发现的问题带到理论知识中去解决，最终把理论与实践融会贯通。 一、概念和常用方法介绍 1、定义用标记的特异性抗体对组织切片或细胞标本中某些化学成分的分布和含量进行组织和细胞原位定性、定位或定量研究，这种技术称为免疫组织化学（immunohistochemistry）技术或免疫细胞化学（immunocytochemistry）技术。 2、原理根据抗原抗体反应和化学显色原理，组织切片或细胞标本中的抗原先和一抗结合，再利用一抗与标记生物素、荧光素等的二抗进行反应，前者再用标记辣根过氧化物酶（HRP）或碱性磷酸酶（AKP）等的抗生物素（如链霉亲和素等）结合，最后通过呈色反应或荧光来显示细胞或组织中化学成分，在光学显微镜或荧光显微镜下可清晰看见细胞内发生的抗原抗体反应产物，从而能够在细胞爬片或组织切片上原位确定某些化学成分的分布和含量。 3、分类 1）按标记物质的种类，如荧光染料、放射性同位素、酶（主要有辣根过氧化物酶和碱性磷酸酶）、铁蛋白、胶体金等，可分为免疫荧光法、放射免疫法、免疫酶标法和免疫金银法等。2）按染色步骤可分为直接法（又称一步法）和间接法（二步、三步或多步法）。与直接法相比，间接法的灵敏度提高了许多。3）按结合方式可分为抗原-抗体结合，如过氧化物酶-抗过氧化物酶（PAP）法；亲和连接，如卵白素-生物素-过氧化物酶复合物（ABC）法、链霉菌抗生物素蛋白-过氧化物酶连结（SP）法等，其中SP法是比较
甲基化DNA测序的试剂配制
Data Supplement Title: Circulating methylated SEPT9 DNA in Plasma is a Biomarker for Colorectal Cancer. Theo deVos1, Reimo Tetzner2, Fabian Model1,3, Gunter Weiss2,Matthias Schuster2, Jürgen Distler2, Kathryn V. Steiger1 , Robert Grützmann5, Christian Pilarsky5, Jens K. Habermann6, Phillip R. Fleshner7, Benton M. Oubre8, Robert Day1, Andrew Z. Sledziewski1, Catherine Lofton-Day1 1) Detailed m SEPT9 Assay Protocol: DNA Extraction: DNA was extracted from 5 mL of blood plasma using a modified viral DNA/RNA extraction kit (chemagen AG, Baesweiler Germany). Plasma samples were thawed at room temperature and extracted following the directions of the kit with modifications. Samples were lysed and treated with protease at 56?C for 10 min in a 50 mL Falcon tube. 100 μL of magnetic particles and 15 mL of binding buffer were then added, and binding was performed for 60 min at room temperature on a rotator. Magnetic particles were captured for 4 min, the supernatant discarded and the pellet was resuspended in 3 mL of wash buffer. 1.5 mL of particle solution were transferred to a 2 mL SafeLock, the beads captured and the supernatant discarded. This was repeated to complete the 3 mL transfer. Tubes were briefly centrifuged and the residual wash buffer was removed by pipetting after bead separation. The tubes were then placed in a 56?C dry block for 5 min, 100 μl of elution buffer was added, the tubes incubated at 65?C with shaking on a thermomixer for 15 min, the particles separated on a magnetic stand and the eluted DNA transferred to a 0.5 mL SafeLock tube (Eppendorf). A 5μl aliquot of the DNA sample was transferred to 45 μl of elution buffer for the measurement of genomic DNA. Bisulfite Conversion: The sample input for bisulfite treatment was 95-100 μl of extracted DNA in elution buffer. The bisulfite reagents (for 25 reactions) were prepared as follows. Bisulfite solution: Sodium bisulfite (4.71gm) and sodium sulfite (1.13gm) were dissolved in 10 mL of ddH2O in a falcon tube, by vigorous shaking and heating to 50?C if required, and the pH adjusted to 5.4-5.5 with 0.2M NaOH as necessary. DME-radical scavenger solution: 188 mg of 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid was dissolved in 1.5 mL diethyleneglycoldimethylether (DME), vortexing to ensure an uniform solution. Bisulfite Reaction: 190 μL of bisulfite solution and 30μl DME-radical scavenger solution were added to the 95-100 μl DNA sample in 0.5 mL SafeLock tubes. The tubes were incubated in a Eppendorf Mastercycler (Eppendorf)according to the following protocol: 5 min 99°C, 25min 50°C, 5 min 99°C, 1h 25min 50°C, 5 min 99°C, 4h 55min 50°C, hold 20°C. This protocol allowed overnight bisulfite conversion. Bisulfite Purification: Following bisulfite conversion, DNA was purified using a customized kit from chemagen AG. The bisulfite reaction (320 μL) was transferred to a 2 mL SafeLock tube, and 1 μLof polyA (500 ng/μL) and 1.5 mL of binding buffer were added. 10 μL chemagen magnetic particles were added and the sample was mixed by vortexing. The samples were incubated at room temperature on a thermal mixer at a rotation of 1000 rpm for 60 min. Magnetic particles were separated on a magnetic stand, the liquid discarded, the tubes briefly centrifuged and the residual liquid removed following magnetic separation. The particles were washed twice with wash buffer II from the kit, and once with 70% ethanol. Following the ethanol wash, the tubes were centrifuged again, and the residual liquid removed following magnetic
免疫组化超详细步骤
免疫组化 一实验目的：分别用KRAS、NRAS、HRAS蛋白抗体检测肺癌组织中KRAS、NRAS、HRAS蛋白的表达情况 二实验原理: 应用免疫学基本原理——抗原抗体反应，即抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂（荧光素、酶、金属离子、同位素）显色来确定组织细胞内抗原（多肽和蛋白质），对其进行定位、定性及定量的研究，称为免疫组织化学技术或免疫细胞化学技术. 三实验试剂及耗材:经病理医生确认的肺癌组织切片、KRAS、NRAS、HRAS蛋白抗体、DAB 显色试剂盒、抗原修复液、PBS、苏木素染料、1%盐酸酒精溶液、0.05%氨水、二甲苯、等 四实验设备：切片机、4℃冰箱、显微镜、烤片机 五主要试剂配置: 缓冲液 应用液A NaH2PO4 38.4g配成1000毫升溶液 应用液B Na2HPO4·12H2O 114.8g配成1000毫升溶液 配制1000毫升PBS需要14毫升应用A液，36毫升应用B液，加8.5gNaCl。 枸橼酸钠抗原修复液 应用液A 枸橼酸 10.55g配成1000毫升溶液 应用液B 柠檬酸三钠（三水合柠檬酸三钠） 29.01g 配成1000毫升溶液 配制500毫升抗原修复液需要9毫升应用A液，41毫升应用B液。 苏木素染液配方：苏木素2g,无水乙醇250毫升，硫酸铝17.6g蒸馏水750毫升，碘酸钠0.2g，冰醋酸20毫升。先将苏木素溶于无水乙醇，再将硫酸铝溶于蒸馏水水中。两液溶解后将其混合，加入碘酸钠，最后加入冰醋酸。 抗体说明书建议的抗体稀释度 N-Ras 1:50-1:500 H-Ras 1:50-1:500 K-Ras 1:20-1:200 六实验步骤： 1组织常规石蜡切片厚度3-5um，防脱片捞片后晾干，放入72℃烤箱烤片2小时。 2 切片脱蜡水化程序
甲基化定量试剂盒与羟甲基化DNA定量试剂盒产品特点--Epigentek
甲基化DNA定量试剂盒与羟甲基化DNA定量试剂盒产品特点 --Epigentek 启维益成提供---甲基化DNA定量试剂盒(比色法) MethylFlash Methylated DNA Quantification Kit (Colorimetric) 产品优点： 1、整个比色法检测实验的操作简单易学，方便快捷，能定量检测全基因组的DNA甲基化 水平，只需要4小时就能完成实验。 2、新颖的试剂盒组分使背景信号极低，去掉了平板封闭并使分析更加地简单、精确、可靠、 稳定。 3、灵敏度高，能在50ng的基因组DNA中检测出低至0.2ng的甲基化DNA。 4、优化的抗体和增强溶液对检测5mC具有高度特异性，不会对未甲基化的胞嘧啶产生交 叉反应，且在指定的样品DNA的浓度范围内与羟甲基胞嘧啶无交叉反应或有轻微的交叉反应 5、试剂盒提供通用阳性和阴性对照，能用于定量检测任何物种来源的甲基化DNA，包括 哺乳动物、植物、真菌、细菌以及病毒； 6、96孔板模式使分析具有灵活性，您可以根据自己的需要选择用手工或者高通量分析。 启维益成提供---甲基化DNA定量试剂盒(荧光法) MethylFlash Methylated DNA Quantification Kit （Fluorometric） 产品优点： 甲基化定量试剂盒荧光法的产品优点与比色法基本相同，不同点为灵敏度不同，比色法能在50ng的基因组DNA中最低检测出0.2ng的甲基化DNA；荧光法能在20ng的基因组DNA中最低检测出50pg的甲基化DNA。 启维益成提供---羟甲基化DNA定量试剂盒(比色法) MethylFlash Hydroxymethylated DNA Quantification Kit (Colorimetric) 关于5-hmC 5-hmC是近年来在动物组织中发现的，由胞嘧啶修饰而来。5-hmC在表观遗传学上的功能可能与5-甲基化胞嘧啶(5-mC)不同，尽管到现在为止还不确知其功能。有研究者猜测它在调控基因的表达与关闭过程中起着重要的作用。5-mC的发现让我们不得不重新评估DNA
DNA甲基化在动植物遗传育种中的研究进展_徐青
doi:10．3969／j．issn．1009－0002．2011．01．026综述DNA甲基化在动植物遗传育种中的研究进展 徐青1，余云舟2，赵萌1，2，孙东晓3 1．北京交通大学生命科学与生物工程研究院，北京100044；2．军事医学科学院生物工程研究所，北京100850；3．中国农业大学动物科技学院，北京100193 ［摘要］DNA甲基化是真核生物表观遗传学重要的机制之一，对基因转录水平的表达具有重要的调控作用。近年来，DNA甲基化在动植物遗传育种领域的研究引起了人们广泛的关注。我们从DNA甲基化与基因的表达调控、动植物基因组的甲基化状态、甲基敏感扩增片段多态性方法、DNA甲基化与杂种优势，以及DNA甲基化与分子标记等方面，简要综述了国内外有关DNA甲基化在动植物遗传育种研究中的进展，着重于全基因组DNA甲基化模式在动植物遗传育种中的相关研究和应用。 ［关键词］DNA甲基化；基因表达；杂种优势；分子标记；基因组 ［中图分类号］Q75［文献标识码］A［文章编号］1009－0002(2011)01－0113－05 Progress of DNA Methylation in Genetics and Breeding of Plant and Animal XU Qing1,YU Yun－Zhou2,ZHAO Meng1,2,SUN Dong－Xiao3 1．Institute of Life Science and Biotechnology,Beijing Jiaotong University,Beijing100044;2．Beijing Institute of Biotechnology,Beijing100071;3．College of Animal Science and Technology,China Agricultural University,Beijing 100094;China ［Abstract］As one of the important epigenetic mechanisms in eukaryotes,DNA methylation plays a crucial role in regulation of gene expression in mRNA level．In recent years,more attention was paid upon DNA methylation in plant and animal genetics and breeding．The progresses in this area were reviewed as following:mechanisms of DNA methylation on regulation of gene expression,DNA methylation patterns in genome of plant and animal, method of methylation sensitive amplification polymorphism,DNA methylation and heterosis,and DNA methylation and molecular marker,with the emphasis on the research and applications of DNA methylation patterns in whole genome in plant and animal genetics and breeding． ［Key words］DNA methylation;gene expression;heterosis;molecular marker;genome DNA甲基化是真核细胞基因组主要的修饰方式之一，同时也是表观遗传学的主要机制之一。近年来，有关表观遗传学的研究取得了突飞猛进的进展，而作为表观遗传学的重要组成部分，DNA甲基化研究已深入到生物、医学、农业及环境等各个领域。我们从DNA甲基化与基因的表达调控、动植物基因组的甲基化状态、DNA甲基化与杂种优势，以及DNA甲基化与分子标记等方面，简要综述了近年来国内外DNA甲基化在动植物遗传育种研究中的新进展，着重于全基组DNA甲基化模式在动植物遗传育种的相关研究和应用。1DNA甲基化与基因的表达调控 1．1DNA甲基化修饰 DNA甲基化是指在各类DNA甲基化转移酶的作用下，将S－腺苷甲硫氨酸（SAM）上的甲基基团共价结合到DNA分子的胞嘧啶碱基或腺嘌呤碱基上的生化过程，它是表观遗传学和表观基因组学的重要研究内容。在哺乳动物中，DNA甲基化包括去甲 ［收稿日期］2010－07－29 ［基金项目］国家高技术研究发展计划重点项目（2007AA10Z157）； 转基因生物新品种培育重大专项(2009ZX08009－156B)； 中央高校基本科研业务费专项（209JBM107）； 北京市自然科学基金（6112018） ［作者简介］徐青（1975－），女，博士，讲师 ［通信作者］孙东晓，（E－mail）sundx＠cau．edu．cn
序列相关例子
问题：1、当设定模型为i i x y εββ++=)ln()ln(10时，是否存在序列相关。 2、若存在序列相关，对原模型进行检验 3、采用差分形式1t * 1* ---=-=t t t Y Y Y X X X 与作为新数据估计模型 t t t X Y εαα++=*10*，该模型是否存在序列相关？ 1模型设定i i x y εββ++=)ln()ln(10 2对原模型做普通最小二乘回归得)ln(854415.0588478.1)?ln(x y += 3回归检验 （1）做残差之间的散点图，观察是否存在序列相关
（2）回归检验法 ....... ~~~~~22111υερερευερε++=+=--t t t t 1>j 检验是否存在一阶序列相关：对模型一做普通最小二乘回归得1766551.0?-=t t e e ，通过T 检验得到 056.2)26(703497.6025.0=>=t t ，则拒绝0=ρ的原假设，说明原模型存在一阶序列相关 2>检验是否存在二阶序列相关：对模型二做普通最小二乘回归得21531439.0191404.1?---=t t t e e e ， 对两个自变量的系数ρ做T 检验得其T 值均大于其临界值则表明原模型存在二阶序列相关 3>检验是否存在三阶序列相关：对模型三做普通最小二乘回归得 221008925.0499919.0222109.1?---+-=t t t t e e e e 对三个自变量的系数i ρ做T 检验得21,ρρ的T 值大于其临界值，3ρ的T 值小于临界值，则说明原模型存在二阶序列相关。 （3）D-W 检验 读取D-W 值0.379323，29.1=l d ，L d W D <<.0，说明原模型存在一阶序列相关。（但其无法检验 模型是否存在二阶序列相关，需要进行拉格朗日法检验） （4）拉格朗日乘数检验（LM ）(View-residual tests-LM test) 1>检验是否存在二阶序列相关：46328.182 =nR >99.5)2(2 05.0=χ则拒绝无序列相关的原假设，且 滞后高阶的T 值大于其临界值，则存在二阶序列相关。 2>检验是否存在三阶序列相关：81.7)3(52001.18205.02 =>=χnR ,则拒绝原假设，表明可能存在3 阶的序列相关。对滞后三阶进行T 检验，069.2)23(394437.0025.0=<=t t ，表明在95%的置信度下 不拒绝原假设0H ，表明变量3-t e 是不显著的，未通过变量的显著性检验，即：原模型存在二阶序列相关。 4序列相关的修正：广义差分法 （1）建立模型i AR AR x y ερρββ++++=)2()1()ln() ln(2110 （2）对该模型进行普通最小二乘估计得： )2(516672.0)1(153100.1)ln(865725.0462413.1)ln(AR AR x y -++= （3）检验该模型是否存在序列相关：读取D-W 值1.819703，表明该模型不存在一阶序列相关，即原模型的修正成功。 5采用差分形式 1t *1*---=-=t t t Y Y Y X X X 与作为新数据估计模型t t t X Y εαα++=*10* （1） 对新模型做普通最小二乘回归得** 596413.03388.889t t X Y +=，读取D-W 值0.960842， 32.1.0=<