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2008 Expression profiling of microRNA using oligo DNA arrays

Expression pro?ling of microRNA using oligo DNA arrays

Chang-Gong Liu

b,1

,Riccardo Spizzo

a,1

,George Adrian Calin a ,Carlo Maria Croce

b,*

Department of Experimental Therapeutics,M.D.Anderson Cancer Center,Houston,TX 77030,United States

Department of Molecular Virology,Immunology and Medical Genetics and Comprehensive Cancer Center,Ohio State University,

Wiseman Hall Room 385,40012th Avenue,Columbus,OH 43210,United States

Accepted 25October 2007

Abstract

After 12years from its ?rst application,microarray technology has become the reference technique to monitor gene expression of thousands of genes in the same experiment.In the past few years an increasing amount of evidence showed the importance of non-coding RNA (ncRNA)in di?erent human diseases.The microRNAs (miRNAs)are one of the groups of ncRNA.They are small RNA frag-ments,19–25nucleotides long,with a main regulatory function on both protein coding genes and non-coding RNAs.The application of microarray platforms applied to miRNA pro?ling determined their deregulation in virtually all human diseases that have been studied.We previously developed a custom miRNA microarray platform,and here we describe the protocol we used to work with it including the oligo design strategy,the microarray printing protocol,the target-probe hybridization and the signal detection.ó2007Elsevier Inc.All rights reserved.

Keywords:microRNA;Microarray;Non-coding RNA;Cancer;Tumor pro?lling;Other diseases

1.Protein-coding genes versus non-coding RNA

Although 5%of the human genome evolves under puri-fying selection,only 2%of this set is represented by protein coding RNA (i.e.protein coding genes)[1].The remaining 3%is split into non-genic DNA and non-coding RNA [2,3].The main di?erences between protein coding and non-coding RNA (mRNA and ncRNA,respectively)is based on the absence of any Open Reading Frame (ORF)for ncRNA and on the di?culty to predict the transcript of ncRNA from its genomic structure [4].The ncRNA fam-ily is heterogeneous,based on the di?erent size of the prod-uct (from few nucleotides to thousands)and on its function.NcRNA can have a generic function such as ribosomal RNA (rRNA)and transfer RNA (tRNA),both involved in mRNA/protein translation,or small nuclear RNA (snRNA)involved in splicing,and ?nally small nucleolar

RNA (snoRNA)involved in the modi?cation of rRNA.The last members of the ncRNA to be discovered are involved in post-transcriptionally regulating protein expres-sion through small RNA molecules such as small interfer-ence RNA (siRNA)[5]and microRNA (miRNA)[6,7].2.Di?erent approaches to quantify tiny RNA molecules The mature miRNAs are very small RNAs,19–25nucle-otides (nt)long and because of this small size there are dif-ferences in the approaches to quantify miRNAs and mRNAs.For example small RNAs are less e?ciently pre-cipitated in ethanol and for this reason during the isolation by standard Trizol protocol of the RNA,resuspension in ethanol should be avoided.On the other hand,in our expe-rience the miRNAs seems to be more stable than longer RNAs,for example in degraded samples it is still possible to obtain readable miRNA expression data.Moreover other groups reported a higher stability of miRNAs com-pared to mRNA in samples obtained from formalin-?xed para?n-embedded tissues [9–11].

Corresponding author.

E-mail address:carlo.croce@https://www.wendangku.net/doc/1c15745344.html, (C.M.Croce).1

These authors contributed equally to this work.

https://www.wendangku.net/doc/1c15745344.html,/locate/ymeth

Available online at https://www.wendangku.net/doc/1c15745344.html,

Methods 44(2008)

22–30

When the?rst miRNAs were described[6,7,12],North-ern blotting was used to detect these small RNAs.The pro-tocol for Northern Blotting was modi?ed to detect such small RNA fragments,a hundred-fold less than the average coding RNA,by using high percentage of urea–acrylamide gels instead of usual agarose gels.

We can classify three main techniques to detect and quantify miRNA in tissue samples.They are the cloning of miRNA,the PCR-based detection,and?nally the hybridization with selective probes.The cloning of miRNA was the main tool used to identify miRNAs[7,8,13,14]. Cloning o?ers the advantage to discover new miRNAs not predicted from bioinformatics and to sequence the miRNAs.On the other hand,the quanti?cation ability is smaller compared to other approaches.The PCR-based technique is able to detect low copy number with high sen-sitivity and speci?city on both the precursor[15,16]and the mature form of miRNAs[17].It is cheap and it can be used extensively for clinical samples with minute amounts of available RNA.The hybridization techniques comprise Northern blotting,in situ hybridization[17,18],bead-based ?ow-cytometry[19]and microarray[32].Northern Blotting using radioactive probes,is very sensitive,but it is very time-consuming.Northern blotting is not practical in large clinical studies to detect the expression of hundreds of miRNAs and it also requires large amounts(5–25l g)of total RNA from each sample.Since the discovery of miR-NAs in Caenorhabditis elegans.

The number of miRNA quickly increased and were found in all the eukaryotic species analyzed[12,20–22].In August 2007,the number of miRNAs has passed500[https://www.wendangku.net/doc/1c15745344.html,/sequences/],and almost a thousand have been predicted in the human genome.Intronic miRNA pre-cursors that bypass Drosha processing have been recently described in Drosophila melanogaster[23,24].These recent discoveries could also potentially expand the number of miRNAs.The increasing number of miRNAs and the anal-ysis of large cohort of patients require a technique able to process multiple miRNAs at the same time and use a rela-tively small amount of RNA obtained from patients.

3.Microarray technology on miRNAs

The microarray technology was developed in1995[25]. It is based on the ability to perform multiple hybridizations in parallel using a glass or quartz support where,depending on the platform used,the probes have been spotted or syn-thesized by photochemical synthesis,[26–28].The ability to increase the density of the spots on the array resulted in a higher number of genes that are able to be analyzed simul-taneously[28,29].Three di?erent technologies classically exist to detect nucleic acids(DNA or RNA)on an array platform.The?rst,commonly used for custom arrays,uses glass slides(poly-lysine coated common microscope slides) and is based on the spotting of unmodi?ed oligonucleotides over the slide[30].The second uses glass slides too,and it is also based on the deposition of the probes on the slide.The distinction is that the50terminus of the probe is cross-linked to the matrix on the glass.The number of probes present on these slides can be much higher compared to the former.In the last technology,the probes are photo-chemically synthesized directly on the surface which is made of quartz.In the last case,the number of probes rises to millions per area the size of a thumbnail[27].Usually, but not always,the?rst two are based on the comparison of two samples for each glass(one used as reference) stained with di?erent colors.The third uses a single color hybridization where each slide is hybridized with only one sample.

The short length and the small abundance of miRNAs determine a more di?cult design of the probes and require a higher sensitivity.Being miRNAs19–25nt long,the design of the probe is almost exclusively determined by the sequence of the miRNA itself,which determines a dif-ferent temperature of annealing for each probe::miRNA interaction.

Since the?rst time array technology was applied to miRNA studies[31,32],we have identi?ed at least18pub-lications describing di?erent approaches for miRNA quan-ti?cation on microarray platforms(Table1).Most of them use DNA oligo spotting,some of them use locked nucleic acid(LNA)with the intent of increasing the a?nity between the probes and the miRNAs,and reaching more uniform conditions of hybridization among the di?erent probes[33].The ideal technique should be able to detect the miRNAs without any kind of manipulation of the sam-ples,such as enrichment of the low molecular weight spe-cies of RNA,retro-transcription of the miRNA, ampli?cation of the miRNA,and in the meantime it should be able to discriminate the two predominant forms of miR-NAs(mature and precursor as it occurs with the array ini-tially developed by us).The di?erences in the expression between mature and precursor forms can represent a signif-icant feature of miRNA biogenesis as it has been proven for two examples,the cluster of mir-143–145and the clus-ter of mir-15a-16.In the?rst example the two miRNAs showed a di?erent expression of the mature form between colon cancer samples and normal colon samples,while no di?erences were found for the precursor forms[34]; while in the second case a mutation of the precursor described in two patients is able to determine a decreased maturation of the miRNA,with a consequent decrease of the mature form[35].

4.Signi?cance of microRNA pro?ling

The miRNAs have been related to the regulation of dif-ferent biological processes.They have been shown to regu-late the C.elegans developmental timing[6,36],as well as the embryonic and post embryonic di?erentiation in rodents[37,38]and in humans[39–41].MiRNAs have been involved in physiological processes,such as insulin regula-tion[42,43],lipid metabolism[44]and synaptic activity, [45].

C.-G.Liu et al./Methods44(2008)22–3023

The?rst evidence of the involvement of microRNA in a human disease was set in2002,when for the?rst time a deregulation of two miRNAs(mir-15a and mir-16-1)was related to Chronic Lymphocytic Leukemia(CLL)[46]. Many other examples followed after that,and many types of tumors were analyzed,including from lung cancer [47,48],breast cancer[49],colon cancer[34,50]and nervous tumors such as glioblastoma[51](for a detailed reference see[52]).The majority of the published papers that reported pro?ling analysis were performed using micro-array technology.The creation of lists of miRNAs di?eren-tially expressed between tumor and normal tissues or between di?erent tumor types,gives the chance to identify the miRNAs most probably involved in cancer and to iden-tify new diagnostic and prognostic markers[35].Since miRNAs are involved in cell di?erentiation[53]and in the in?ammatory response[54,55],their deregulation in cancer can be just a consequence of the phenotypic changes in the tumor cells and in the surrounding normal tissues due to the cancer development,and consequently the deregulated miRNAs could not play an active role in the tumor pathogenesis.Consequently,to prove a causative role of a speci?c miRNA in a tumor pathology,functional studies are mandatory and they have to link the miRNA function with cellular process such as proliferation[56], apoptosis[57,58],and metastasis/migration[59].A de?nite proof for the causal link between miRNAs and cancer is the development of B cell malignant proliferation in a miR-155transgenic mouse model[60].

The leading cause of death in western countries still remains heart diseases[61].Recent data report that mir-1is involved in the regulation of the cardiogenesis and cardiac conduction in rodents[38].A previous study iden-ti?ed the miRNA signature of cardiac hypertrophy in rodents,and in another study the transgenic mouse model of one of the described upregulated miRNAs(mir-195) was able to reproduce a phenotype close to the disease [62].Among the neurologic diseases,Alzheimer’s disease is the most frequent cause of death in the US[61]and its etiology remains unexplained.The miRNAs are involved in di?erent brain functions too.One of the?rst examples of miRNA pro?ling studies showed a speci?c signature made of44miRNAs during the rodent brain development[31].A study correlated the deregulation of miRNAs in the hippocampus of patients a?ected by Alz-heimer’s disease[63].Among the most frequently involved miRNAs in brain development and pathology are mir-9, mir-125b(the orthologous of lin-4the?rst miRNA dis-covered in C.elegans[6])and mir-128.Functional studies and mouse models relating miRNA deregulation and brain diseases are still missing,but the pro?ling studies showed the?rst evidence of the potential involvement of miRNAs on brain pathologies.

The discovery of miRNAs de?ned a completely new reg-ulatory system.For this reason these small RNA molecules can help to?nd some of the hidden etiologic mechanisms for some diseases.Furthermore the better understanding of the etiology and the pathogenesis of a disease can create new diagnostic tools and therapeutic targets.Screening the expression of all miRNAs by cDNA microarray at once,is in our opinion,the?rst step in the study of the involvement of miRNAs in one speci?c pathology.The di?erential expression between di?erent groups becomes the main cri-teria on microarray analysis and it can suggest that further studies on particular miRNAs that can successfully show a direct involvement in the etiology and/or pathogenesis of that disease.In the meantime the use of microarray plat-forms is probably the best way to study the existence of completely new transcribed genomic elements and to deter-mine their tissue speci?city and possible involvement in dif-ferent diseases such as cancer[64].

In order to pro?le miRNA gene expression and to obtain genome wide signatures in diseases such as cancer, we developed a unique miRNA microarray using a Code-Link platform(https://www.wendangku.net/doc/1c15745344.html,/microarray). We will focus on the equipment,supplies,and protocol that are necessary to develop a microarray platform start-ing from the oligo design,through the array fabrication and miRNA target preparation,to the hybridization of the samples and the signal detection and data analysis (Fig.1).

5.Discussion of the equipment(for list of instruments, supplies and reagents see Appendix)

The Ohio State University Comprehensive Cancer Cen-ter(OSUCCC)miRNA Expression Bioarrays are tran-scriptional pro?ling products designed to monitor the miRNA and other small non-coding RNA levels of multi-ple genes.The miRNA Expression Bioarrays utilize nucleic acid hybridization of a50biotin-labeled complementary cDNA target with DNA oligonucleotide probes attached to a gel matrix.

The biotin-labeled cDNA targets are prepared by a sim-ple reverse transcription into?rst strand cDNA.Total RNA is primed for reverse transcription by a random Octomer conjugated with two biotins and a50poly(A)tail. This procedure results in an equal copy number of biotin–cDNA target to the templates of miRNA.

5.1.Storage and handling conditions

These procedures involve working with RNA;therefore, care must be taken to avoid any potential RNase contam-ination.All solutions must be RNase-free and pipette tips must be changed before each https://www.wendangku.net/doc/1c15745344.html,e commercially pre-pared nuclease-free H2O(in lieu of H2O treated with diethylpyrocarbonate)for all nucleic acid steps.

Store the cDNA synthesis kits,except for the hybridiza-tion bu?er,atà20°C(do not store in frost-free freezer).

Store the hybridization bu?er components as indicated on the tubes or bottles.

The Invitrogen’s Trizol product is recommended for total RNA isolation.No column procedure should be

C.-G.Liu et al./Methods44(2008)22–3025

applied for total RNA isolation or cleaning.miRNA will be lost when a column is applied to total RNA isolation.Any Trizol contamination in total RNA can kill the enzyme in the target labeling reaction.

5.2.MiRNA microarray fabrication

5.2.1.Oligo design

Two 40mer oligo probes,one for the mature miRNA and the other for precursor oligo,were designed from the sense strand of both arms of the hairpin structure of the microRNA precursor sequence collected from the Sanger Database (https://www.wendangku.net/doc/1c15745344.html,/cgi-bin/sequences/browse.pl ).The oligo probes were modi?ed at the 50end with Amine-C6linker and ordered from Integrated DNA technology (IDT)(Coralville,IA,USA)at 50or 100l M stock concentration in H 2O.

5.2.2.Oligo probe library for miRNA array fabrication The working oligo library should be in the concentration of 20l M in 50mM Sodium Phosphate bu?er pH 8.0with 2l M of Sodium-Fluorescein.The oligos were synthesized in a 96well plate format from the vendor,and we had to assemble in house four 96well plates into 384well microti-ter plate format by Tecan TeMo liquid handler in order to be able to work with the array fabrication system.For qual-ity control (QC)purpose of chip fabrication,the oligo probes are co-dispensed with Fluorescein (Sigma F-6377).5.2.3.miRNA array fabrication

The Codelink ?activated slides (GE Healthcare,USA,PN 300011)are loaded onto an Omnigrid 100slide holder.The 384well oligo library plates are loaded sequentially onto a Gene Machine OmniGrid 100arrayer (Genomic Solution,Ann Arbor,MI,USA)and the miRNA arrays are printed with a designed printing protocol (Reference,Omnigrid user manual website).

5.2.4.miRNA array post-printing processes

5.2.4.1.Quality control scanning.The printed array slides were scanned by Axon Scanner 4200at 488nm excitation length in order to detect ?uorescein,and QC image ?les were saved.

5.2.4.2.Oligo probe coupling.Under 70%humidity over-night,the dispensed amine-modi?ed oligo probes on the slides couple with NHS ester group of coated polymer gel matrix and are covalently immobilized on the surface of Codelink ?activated slides.

5.2.4.3.miRNA array blocking.The NHS ester groups sur-rounding the spots of oligo probe dispensed need to be blocked,in a New Brunswick Innova 4080Shaking incuba-tor,in a solution of 50°C prewarmed 100mM Bicine (Sigma B-8660)and 100mM Taurine (Sigma T8691),pH 9.0at 50°C for 60min.Then the blocked slides are rinsed with deionized H 2O and further washed in 50°C pre-warmed 4·SSC/0.1%SDS bu?er for 30min with 50rpm agitation.The slides were rinsed with H 2O and spun dry.The slides were now ready to hybridize the labeled miRNA cDNA targets and should be stored in a desiccator (Fisher 08-642-23C)until

use.

Fig. 1.Principles of microarray technology used for microRNAs pro?ling.(A)The microarray based miRNA pro?ling is presented as described in the majority of pro?ling studies on primary tumors,initially developed by Liu et al.[32].Target labeling,hybridization,staining and signal detection are the four main technical steps (presented on the right side).The di?erent replicates of the spots on the glass slide (presented as di?erent types of gray grids)represent di?erent oligonucleotide sequences corresponding to sequences from the precursor miRNA or active miRNA molecule.The main advantage of the microarray based miRNA pro?ling is the high standardization of the procedure (allowing the processing of tens of samples in parallel),making it relatively easy to be performed.Several technical aspects regarding this technology can be found elsewhere [79].Reproduced after Calin and Croce [52].

26 C.-G.Liu et al./Methods 44(2008)22–30

5.3.Sample preparation

5.3.1.Synthesis of biotin labeled?rst-strand cDNA targets (1)Prepare each total RNA sample for manual target

preparation

5l g Total RNA(optimal concentration should be determined for each source of total RNA).

2l l0.5l g/l l primer.

X l l Nuclease-free H2O to12l l?nal volume.

12l l Total volume.

[5l g of total RNA in10l l RNAse free H2O

2l l of oligo primer(50biotin-AAA-AAA-AAA-AAA-(T-biotin)-AAA-AAA-AAA-AAA-NNN-NNN-NN30) (0.5l g/l l)(where N stands for random octamer).] (2)Incubate10min in a70°C water bath then immedi-

ately place the tube on ice.

(3)Centrifuge for5s to collect the sample at the bottom

of the tube and immediately place the tube on ice.

(4)With the tube remaining on ice,add the following

reagents to the12l l of total RNA/control mRNA/ primer mix.

4l l5·?rst-strand bu?er.

2l l0.1M DTT.

1l l10mM dNTP mix.

1l l Superscript?II RNaseHàreverse transcriptase (200U/l l).

20l l Final volume

(5)Incubate90min in a37°C water bath.

(6)Centrifuge for5s to collect the sample at the bottom

of the tube.

(7)RNA template degradation.After a90min incubation

for the?rst strand synthesis,adding3.5l l of0.5M NaOH/50mM EDTA into20l l reaction mix and incubate at65°C for15min to denature the DNA/ RNA hybrids and degrade the RNA template.Then neutralize the reaction with5l l of1M Tris–HCI, pH7.6(Sigma).Each labeled target should be in a vol-ume of28.5l l.The sample preparation is now done and the samples are stored atà80°C until use.

5.4.miRNA microarray hybridization and data collection 5.4.1.miRNA microarray hybridization

(1)Prime all channels of the Tecan HS4800hybridiza-

tion stations and load hybridization chambers to HS4800.

(2)Load pre-printed miRNA array face-up to the Tecan

HS4800hybridization station.Close hybridization chambers on HS4800.

(3)Run hybridization program for the chip hybridiza-

tion with labeled cDNA targets:

a.Prime the chip in the hybridization chamber at23°C

with6·SSPE with0.05%Tween20for1min.

b.Inject75l l prehybridization mix of6·SSPE/2·

Dehardt/30%formamide and prehybridize the chip at 25°C for30min.

c.Inject the hybridization mix of labeled biotin–cDNA

in6·SSPE/2·Dehardt/30%Formamide and hybridize at25°C for18h.

d.Wash in0.75·TNT bu?er at23°C for5min.

e.Wash in0.75·TNT bu?er at37°C for10min.

f.Water rinse on the HS4800at23°C for30s.

g.Unload the chips from the machine for post-hybrid-

ization washing.

5.4.2.miRNA microarray post-hybridization

(1)Open the hybridization chamber of the HS4800and

remove the slide chips as quickly as possible and then place them into a slot of the Bioarray Rack,which was placed in the large Reagent Reservoir containing 37°C prewarmed0.75·TNT.Move the slide into place using the Bioarray Position Tool,tooth-side down.Wash them in37°C prewarmed0.75·TNT with agitation in New Brunswick Innova4080Shak-ing incubator at37°C for40min with50rpm agitation.

(2)Block the chip in TNB blocking bu?er(see Appendix)

at room temperature for30min.

(3)Stain the chips with streptavidAlexa-6471:500in

TNB bu?er at room temperature for30min.

(4)Post-stain wash in1·TNT at room temperature for

40min in total with three bu?er changes.

(5)Rinse the chips with distilled water brie?y and spin

dry them at1000rpm for1min.

5.4.3.miRNA microarray data collection

Scan the chip by Axon Scanner at a power setting of Power100and PMT800.The image data may be extracted by GenePix software and saved as a.gpr?le for further data analysis.

Acknowledgments

Dr.Calin is supported by the CLL Global Research Foundation,and,in part,as a University of Texas System Regents Research Scholar and as a Fellow of The Univer-sity of Texas M.D.Anderson Research Trust and Dr. Croce is supported by Program Project Grants from the National Cancer Institute.We apologize to our colleagues whose work was not cited due to space limitations.

C.-G.Liu et al./Methods44(2008)22–3027

Appendix A.Appendixes(solutions,equipment,and supplies)

A.1.MiRNA microarray fabrication(see Sections5.2.1–

5.2.4)

A.1.1.Solutions for miRNA array fabrication

(1)One hundred micromolar phosphate bu?er pH8.0

(2·):Resuspend0.69g of anhydrous monobasic sodium phosphate(Sigma S-3139)and25.46g of diba-sic sodium phosphate,heptahydrate(Sigma S-9390)in 900ml of distilled H2O and adjust the solution pH to

8.0by adding100l l of10N NaOH.Add more distilled

H2O to1000ml volume and?lter the solution by

0.22l m?lter unit.The100mM(2·)phosphate bu?er

is stored at4°C until use.

(2)Two micromolar?uorescein solution:First prepare a

200l M solution by dissolving18.845mg of Fluores-cein(Sigma F-6377)in250ml of H2O into as200l M solution?rst.Dilute5ml of200l M solution into 495ml of H2O(2l M Fluorescein solution).

(3)One hundred micromolar bicine and taurine array

blocking solution:Dissolve48.9g of Bicine(Sigma B-8660)and37.5g of Taurine(Sigma T-8691)in2400ml of H2O.Adjust the pH to9.0by adding$40ml of 10N NaOH.Add more H2O to a volume of3000ml.

(4)4.04·SSC/0.1%SDS array washing solution:Make

4.04·SSC from20·SSC solution(Sigma S-6639)

by dilution?rst.Mix together2970ml of4.04·SSC and30ml of10%SDS.

A.1.2.Equipment and materials

?Omnigrid100Arrayer:Genomic Solution,Inc.4355 Varsity Dr.Suite E,Ann Arbor MI48108.

?Tecan TeMo liquid handler:Tecan TEMO liquid han-dler,TECAN US Inc.Research Trangle Park, NC27709.

?Axon Scanner4200:Molecular Device Corp.1311 Orleans Dr.Sunnyvale CA94089-1136.

?CodeLink Activated Slides:GE Healthcare(Amersham-300011),Piscataway,NJ.

?20·SSC Sigma S6639-1L.

?5M Sodium Chloride,Sigma S5150-1L.

?Bicine,Sigma B8660-1KG.

?Taurine,Sigma T8691-100G.

?Fluorescein,Sigma F-6377.

?Anhydrous monobasic sodium phosphate,Sigma S-3139.?Dibasic sodium phosphate,heptahydrate,Sigma S-9390.

A.2.Sample preparation(see Section5.3)

A.2.1.Reagents

?0.5l g/l l30NNNNNNNN-(dA)12T(biotin)(dA)12-Biotin50Oligonucleotide primer.

?5·First-strand bu?er.

?0.1M Dithiothreitol(DTT).

?10mM dNTP mix.

?Superscript?II RNaseHàreverse transcriptase(200U/ l l)(Invitrogen18064-014).

?10mM dNTP mix(Invitrogen18427-013).

A.2.2.Equipment and materials supplied by user ?Pipette tips,sterile,RNase-free,and aerosol-resistant.?Microcentrifuge tubes,sterile,RNase-free,1.7ml.?Micropipettes(10,20,200,1000l l).

?Nanodrop UV spectrophotometer.?Microcentrifuge,room temperature and4°C.?Water bath(settings70°C,65°C,37°C).

?Sterile,nuclease-free conical tubes(15and50ml).?Speed-Vac concentrator.

?Vortex.

?Pipette aid and disposable pipetts.

A.3.miRNA microarray hybridization and data collection (see Section A.3.3)

A.3.1.miRNA microarray hybridization

(a)Required reagents/kits supplied by user

?0.5%NEN Blocking Reagent(Perkin Elmer No.FP1020).?Molecular Probes Streptavidin-Alexa Fluorò647conju-gate(staining solution is a1:500dilution in TNB;see Appendix A)(Molecular Probes No.S-21374).?Nuclease-free H2O(Ambion No.9915G).?1·PBS;pH7.4(Invitrogen/LTI No.10010-023).

?1M Tris–HCl,pH7.6(Sigma No.T-2788).

?5M NaCl(Sigma No.S-5150).

?Tweenò-20(Sigma No.P-7949).

?Formamide(Sigma).

?50·Denhardt Solution(Sigma).

(b)Other equipment and materials supplied by user ?Tecan HS4800Hybridization Station.

?Axon GenePix4000B scanner.

?Computer con?gured for Axon4000B Scanner.?New Brunswick Innova?4080shaking incubator.?Sigma/Qiagen Centrifuge(4°C–15°C)(Qiagen No.81010).?Centrifuge Plate Rotor-2x96(Qiagen No.81031).?Pipette tips,sterile,RNase-free,and aerosol-resistant.?Microcentrifuge tubes,sterile,RNase-free,1.7ml.?Micropipettes.

?Powder-free gloves.

?Microcentrifuge.

?Microtiter plate lid,Black(Corning,#3935).?Bioarray Processors(GE,Healthcare).

?Bioarray Rack(GE No.600010).

28 C.-G.Liu et al./Methods44(2008)22–30

?Small Reagent Reservoir(GE No.600011).

?Large Reagent Reservoir(GE No.600013).?Bioarray Removal Tool(GE No.600015).

?Bioarray Position Tool(GE No.600016).

A.3.2.miRNA microarray post-hybridization

(a)Stock solutions for post-hybridization array process-ing TNT bu?er(20L)

0.1M Tris–HCl,pH7.6.

0.15M NaCl.

0.05%Tween-20.

Rinse a25L Carboy out with150ml of isopropanol. Rinse the carboy twice with3L of deionized H2O and completely drain the carboy.

Add2L1M Tris–HCl.

Add600ml5M NaCl.

Add10ml Tween-20.

Add17.39L deionized H2O.Mix well by swirling. Filter TNT through a0.2l m?lter.

This solution can be stored up to2weeks at room temperature.

(b)0.75·TNT bu?er

Add25ml of deionized water to75ml of TNT bu?er (from above)per100ml of bu?er required

(c)TNB bu?er(0.5L).

0.1M Tris–HCl,pH7.6.

0.15M NaCl.

0.5%NEN blocking reagent(Perkin Elmer FP1020). Add435ml nuclease-free H2O.

Add50ml of1M Tris–HCl,pH7.6.

Add15ml of5M NaCl.

Slowly add 2.5g of NEN Blocking reagent in0.5g

increments until all 2.5g of blocking reagent are

dissolved,while warming in a water bath at60°C. Filter TNB bu?er through a0.88l m?lter.

Aliquot the TNB bu?er to50ml tubes and store atà20°C.

This solution can be stored for up to12weeks at à20°C.Thaw immediately before use.

A.3.3.miRNA microarray data collection

Ideally,the same raw data should be analyzed by two distinct bioinformatics using two independent methods of analyses.For a detailed description,see Ref.[52].

Appendix B.Safety precautions

Standard laboratory safety procedures should be fol-lowed when using this product.Safety glasses,a lab coat,and appropriate gloves should be worn at all times when in the laboratory.Additional care should be taken when using Qiagen Bu?er RLT,which contains chaotropic salts. References

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总说明一、辽宁省建设工程计价依据《辽宁省建设工程费用标准》（以下简称费用标准）是工程量清单计价、编制招标控制价或拦标价的依据；是编制建设工程估算、设计概算、施工图预算、竣工结算的依据；是调解处理工程造价纠纷、鉴定工程造价的依据；是投标报价和衡量投标报价合理性的基础；是编制投资估算指标、概算（定额）指标的基础。二、本费用标准与2008年辽宁省建设工程计价依据《A建筑工程计价定额》、《B装饰装修工程计价定额》、《C安装工程计价定额》《D市政工程计价定额》及《E园林绿化工程计价定额》配套使用。三、本费用标准中的企业管理费、安全文明施工措施费，投标人在投标报价时，可根据企业管理水平和工程实际适当调整，但不得低于本费用标准的90%。四、本费用标准的规费，按核定的施工企业规费计取标准执行。五、工程量清单计价中的综合单价，应按本费用标准的“工程费用取费程序表”中的管理费费和利润计算方法并考虑风险组成综合单价。六、使用1996年《全国统一房屋修缮工程预算定额辽宁省单位估价表》、1993年《全国统一市政工程预算定额辽宁省单位估计表（市政维修）》、1988年《全国统一仿古建筑预算定额》的工程，可按2008年相应计价的人工、材料、机械单价进行换算，按照本费用标准相应标准计取各项费用。七、2008年辽宁省建设工程计价依据计价定额材料费中不包括材料检验试验费。招标工程由投标人人在投标报价时自行确定；非招标工程，工程结算时按实际发生计入其他项目费。八、本费用标准中的“xxx以上”，“不包括xxx本身”“xxx以下”，包括xxx本身。第一部分建设工程费用组成根据建设部、财政部建标2003206号文件《关于印发<建筑安装工程费用项目组成>的通知》精神，结合我省实际情况，确定建设工程费用项目组成如下：建设工程费用由直接费、间接费、利润和税金组成。一、直接费直接费由计价定额分部分项工程费和措施项目费组成。（一）计价定额分部分项工程费：由直接工程费和技术措施费组成。直接工程费：是指施工过程中耗费的构成工程实体的各项费用，包括人工费、材料费、施工机械使用费。 1.人工费：是指直接从事建筑安装工程施工的生产工人开支的各项费用，内容包括：（1）基本工资：是指发给生产工人的基本工资。（2）工资性津贴：是指按规定标准发放的物价补贴，煤、燃气补贴，交通补贴，住房补贴，流动施工津贴等。（3）生产工人辅助工资：是指生产工人年有效施工天数以外非作业天数的工资，包括职工学习、培训期间的工资，调动工作、探亲、休假期间的工资，因气候影响的停工工资，女工哺乳时间的工资，病假在六个月内的工资及产、婚、丧假期的工资。（4）职工福利费：是指按规定标准计提的职工福利费。（5）生产工人劳动保护费：是指按规定标准发放的劳动保护用品的购置费及修理费,徒工服装补贴，防暑降温费，在有碍身体健康环境中施工的保健费等。 2.材料费：是指施工过程中耗费的构成工程实体的原材料、辅助材料、构配件、零件、半成品的费用。内容包括：（1）材料原价（或供应价格）

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(韩庚)画意诗情带笑意只为等待你 (汪峰)北京欢迎你像音乐感动你 (莫文蔚)让我们都加油去超越自己 (谭晶)北京欢迎你有梦想谁都了不起 (陈奕迅)有勇气就会有奇迹 (阎维文)北京欢迎你为你开天辟地 (戴玉强)流动中的魅力充满着朝气 (王霞．李双松)北京欢迎你在太阳下分享呼吸 (廖昌永)在黄土地刷新成绩 (林依轮)北京欢迎你像音乐感动你 (张娜拉)让我们都加油去超越自己 (林俊杰)北京欢迎你有梦想谁都了不起 (阿杜)有勇气就会有奇迹京剧：北京欢迎你呀 (容祖儿)我家大门常打开开放怀抱等你 (李宇春)拥抱过就有了默契你会爱上这里 (黄大炜)不管远近都是客人请不用客气 (陈坤)相约好了再一起我们欢迎你 (谢霆锋)北京欢迎你为你开天辟地 (韩磊)流动中的魅力充满着朝气 (徐若瑄)北京欢迎你在太阳下分享呼吸 (费翔)在黄土地刷新成绩

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6.1 其他项目清单与计价汇总表：表－10 6.1.1 暂列金额明细表：表－10－1 6.1.2 材料暂估单价表：表－10－2 6.1.3 专业工程暂估价表：表－10－3 6.1.4 计日工表：表－10－4 6.1.5 总承包服务费计价表：表－10－5 6.2 索赔与现场签证计价汇总表：表－11 6.2.1 费用索赔申请（核准）表：表－11—1 6.2.2 现场签证表：表－11—2 7 人工、材料、机械价格表 7.1 主要工日价格表：表－12 7.2 主要材料价格表：表－13 7.3 主要机械台班价格表：表－14 8 安全防护、文明施工措施项目费分析表：表－15 9 工程款支付申请（核准）表：表－16

2008定额取费标准

建筑、安装、市政、装饰装修工程及其包工不包料工程费用标准一、建筑工程费用标准 (一)费用标准适用范围 1．一般建筑工程费用标准：适用于工业与民用的新建、改建、扩建的各类建筑物、构筑物、厂区道路等建筑工程。 2．建筑工程土石方、建筑物超高、垂直运输、特大型机械场外运输及一次安拆费用标准：适用于工业与民用建筑工程的土石方（含厂区道路土方）、建筑物超高、垂直运输、特大型机械场外运输及一次安拆等工程项目。 3．桩基础工程费用标准：适用于工业与民用建筑工程中现场灌注桩和预制桩的工程项目。 (二)建筑工程费用标准(包工包料) 1.一般建筑工程费用标准(见下表)。 2.建筑工程土石方、建筑物超高、垂直运输、特大型机械场外运输及一次安拆费用标准(见下表) 3.桩基础工程费用标准（见下表）

（三）建筑工程类别划分及说明 1.一般建筑工程类别划分（见下表） 2．桩基础工程类别划分标准。（1）现场混凝土灌注桩为桩基础一类工程。（2）预制混凝土桩为桩基础二类工程。 3．接层工程的工程类别划分：在计算檐口高度和层数时，连同原建筑物一并计算。 4．斜通廊以最高檐口高度，按单层厂房标准划分。 (四)工程类别使用说明 1．以单位工程为类别划分单位，在同一类别工程中有几个特征时，凡符合其中之一者，即为该类工程。 2．一个单位工程有几种工程类型组成时，符合其中较高工程类别指标部分的面积若不低于工程总面积的50%,该工程可全部按该指标确定工程类别;若低于50%,但该部分面积又大于1500㎡，则可按其不同工程类别分别计算。 3．高度系指从设计室外地面标高至檐口滴水的高度（有女儿墙的算至女儿墙顶面标高）。4．跨度系指结构设计定位轴线的距离，多跨建筑物按主跨的跨度划分工程类别。

帕萨特领驭故障两例

汽车维修技师·2012 年第2期 63 故障两例案例1：左右后视镜不能调节行驶里程：210000km。故障现象：一辆2006款帕萨特领驭1.8T手动尊贵版轿车，左右两侧后视镜无法工作。据车主称，一个月前该车曾更换过后视镜开关，要求索赔维修。故障诊断：查阅维修档案证实了车主描述，维修人员根据索赔单，领出新的后视镜开关换上，但遗憾的是故障没能排除。至此，笔者介入故障检修。该车的舒适系统属CAN-BUS结构，采用CAN数据总线的舒适系统，后视镜控制的流程是控制单元接收与识别出开关信号，输出执行指令控制调节电机工作，或向数据总线发出信息，让另一控制单元接收并输出执行指令。帕萨特领驭后视镜控制电路（CAN总线）如图1所示，后视镜左右转换开关E48与调节开关E43的信号发送对象是左前车门控制单元J386，E43和E48均只有一条信号线与 J386相连，开关触点控制接地。E43内部并联4个阻值不同（其中一个为0）的电阻，在J386的T29a/3端子预置12V 电压的条件下，通过开关接入不同的电阻形成相应的电位，J386可以识别出水平调节（X+、X-）与垂直调节（Y+、Y-）的请求信号。同理，J386根据E48两个阻值不同的电阻形成的电位，可以识别出调节左侧或右侧后视镜的请求信号。需要调节左侧后视镜时，J386直接输出指令，执行电流流过左侧垂直或水平调节电机V17或V149，需要调节右侧后视镜时，J386通过CAN数据总线将请求信息传输到右前车门控制单元J387，J387接收识别后输出指令，执行电流流过右侧垂直或水平调节电机V25与V150。检查该车舒适系统的其他功能如电动窗、中控锁与室内灯等均无异常，连接VAS5052诊断仪查询舒适系统控制单元J393，无故障记忆，这些细节都说明舒适系统供电没有问题。开启车灯开关至小灯挡，新换开关上的背景灯没有点亮。点击测量值功能选项，读取3组的测量值为未操作、未操作、未安装、断开，2区是后视镜转换开关E48的信号示值，将 E48置于左侧位置，2区出现向后移动的示值，然后恢复到未操作的显示；置于右侧位置，未帕萨特领驭浙江/ 陈中泽号线都有了12V的电压，空调压缩机可以正常的吸合。到此时我们基本上可以确定故障的原因就是由蒸发箱温度传感器引起的。为了进一步证明，我们向蒸发箱加热，当蒸发箱周边的实际温度已经为20℃时（蒸发箱温度传感器的电阻应该是5k ?左右），蒸发箱表面温度传感器的电阻却只变化到9.5k ?，这说明该传感器的灵敏度已经很差。按照该车的VIN码定了一个蒸发箱表面温度传感器（如图4所示），更换后故障排除（该款车型更换蒸发箱表面温度传感器时需要将仪表台拆下，分解蒸发箱总成后才可以换上）。图4 新的蒸发箱温度传感器图1 帕萨特领驭轿车后视镜开关（CAN总线）电路图

北京欢迎你

北京欢迎你知识链接《北京欢迎你》是杨澜作为北京申奥代表团的成员在申办2008年奥运会时陈述词，文章以饱满的热情，朴实流畅的文字向世界各国朋友介绍了北京的人文环境和地理优势，是举办奥运会最理想的城市，并真诚的欢迎各国朋友来北京旅游观光。教学前，要求学生动手查找有关北京奥运会的资料，了解奥运会的有关知识。教学中，要让学生在充分读的基础上，了解文章从哪些方面阐述了北京是一个举办奥运会最理想的城市之一，并从中感到无比高兴和自豪。学习目标： 1、学习10个生字，了解应用文的基本格式。 2、理解课文内容，抓住重点词句，体会作者充满激情的演讲所表达的思想感情。 3、学习有理有据，条理清楚的表达自己的意思。 4、有感情地朗读课文，激发学生热爱祖国，热爱北京的思想感情。并说说自己将为3008年奥运会的到来做些什么。教学重点：能结合重点词句，理解作者在文章中陈述了哪些内容，体会陈述词为申奥成功所表达出的真切情意和为北京而骄傲的思想感情。教学难点：理解“新的局面”、“新的高度”的含义，学习有理有据、条理清楚地表达自己的意思。教学时间：2课时教学过程：第一课时一、引言激取，导入新课。 1、奥运会是竞技体育。奥运精神，是顽强拼搏，积极进取；是勇于奉献，甘于寂寞；是更快更高更强，自信自强自尊！参加奥运，取胜奥运，是多少人的梦想，我们都一一实现并取得了骄人的成绩，中国在历届奥运会上取得的金牌总数高达112枚！“中国”这个响亮的名字响彻了全世界！东方的巨龙正在腾飞！举办奥运会，更是我们多年的努力、期盼与渴望，这一天终于来到了！2001年7月13日，是每个中国人都难以忘怀的日子——我们的梦想终于实现了！今天，让我们走近那个令人难忘的日子，聆听申奥代表团的代表杨澜的申奥陈述吧！二、自主学习，整体感知。 1、认真地读课文，把课文读通顺，读正确，没有读好的地方可以反复多练练。 2、自学生字新词。 3、看看课文陈述了哪些内容？每个内容能用一句话概括吗？（中国拥有自己的体育传统；北京有许多精彩纷呈的事情和友善的人民；我们的文化计划。）小结：看来，整个陈述词是条理清楚，有详有略地按总分总的顺序陈述，我们读起来感到很清晰。

《建设工程工程量清单计价规范》(GB50500-2008)

附件1 《建设工程工程量清单计价规范》（GB50500-2008）浙江省补充条款一、采用工程量清单计价方式的工程建设项目，除应执行《建设工程工程量清单计价规范》GB50500-2008，还应执行本省的补充规定。二、工程造价文件的编制与核对，如由造价员承担时，应具备相应专业的造价员资格证书。建设项目各方应拒绝未按规定签字盖章的工程造价文件。三、招标人委托工程造价咨询人或招标代理机构编制工程量清单，其编制工作时间应根据工程建设项目的实际情况，在保证编制质量的前提下由双方在咨询合同中协商约定。四、工程造价咨询人或招标代理机构及其工程造价专业人员应对其编制的工程量清单（或招标控制价）质量负责，并在委托合同中约定相关责任条款。五、编制工程量清单出现附录中未包括的项目，编制人应作补充，并在所属的分部列项。六、措施项目清单编制应体现工程特点和招标要求，需补充的措施项目，清单编制人可参照省建设行政主管部门颁发的计价依据补充或自行补充。文明施工与环境保护、临时设施、安全施工是必须保证的措施项目，投标报价时应根据招标文件要求提供分析表。具体编制可参照安全防护、文明施工措施项目分析表，并结合招标工程的特点列项。建设工程检验试验费、建筑工程超高施工增加费作为措施项目，分别列入组织或技术措施项目清单。七、可以计算工程量的措施项目清单，在确定项目名称、特征描述、计量单位和工程量计算规则等时，应满足投标人按本省计价依据进行报价的需要。八、专业工程暂估价一般以“项”为单位编制。暂估价材料如遇本省计价依据中无相类似的材料时，投标人应该在投标文件中明确该暂估价材料的损耗率。九、规费项目由规费1、规费2、规费3组成，具体内容如下： 1、规费1，包括：（1）工程排污费；（2）社会保障费：包括养老保险费、失业保险费、医疗保险费；

北京欢迎你英文版歌词

北京欢迎你 Welcome to Beijing 迎接另一个晨曦带来全新空气 Another morning is coming and brings in the fresh air. 气息改变情味不变茶香飘满情谊 Our friendship will never change, just like the fragrance of green tea. 我家大门常打开开放怀抱等你 My door is always open for you, and I am here for you with open arms. 拥抱过就有了默契你会爱上这里 You’d know me better when you hugged me, and you will love it here. 不管远近都是客人请不用客气 Be my guest no matter you are from home and abroad. 相约好了在一起我们欢迎你 You are here as promised, friends we welcome you. 我家种著万年青开放每段传奇 I grow green plant at home to show you all the legends in the past. 为传统的土壤播种为你留下回忆 Want to plant in the soil to make you good memories. 陌生熟悉都是客人请不用拘礼 Friends and strangers, you are all my guests, and please be at home. 第几次来没关系有太多话题 No matter how many times we meet, there are still a lot to talk about. 北京欢迎你为你开天辟地 Welcome to Beijing, we will show you a brand new place. 流动中的魅力充满著朝气 The charm of Beijing is full of life. 北京欢迎你在太阳下分享呼吸 Welcome to Beijing, let’s breathe together in the sunshine. 在黄土地刷新成绩 We want to make new record on this yellow soil. 我家大门常打开开怀容纳天地岁月绽放青春笑容迎接这个日期天大地大都是朋友请不用客气画意诗情带笑意只为等待你 My doors are always open, we've space for the world Time flies with youthful smiles, we all wait for this day Under the sky, everyone be our guest, so just be at home Nice scenary with smiling faces, all awaiting there for you

2008清单计算规则

2008清单计算规则一、平整场地：建筑物场地厚度在±30cm以内的挖、填、运、找平。 1、平整场地计算规则（1）清单规则：按设计图示尺寸以建筑物首层面积计算。（2）定额规则：按设计图示尺寸以建筑物外墙外边线每边各加2米以平方米面积计算。 2、平整场地计算公式 S=（A+4）×（B+4）=S底+2L外+16 式中：S———平整场地工程量；A———建筑物长度方向外墙外边线长度；B———建筑物宽度方向外墙外边线长度；S底———建筑物底层建筑面积；L外———建筑物外墙外边线周长。该公式适用于任何由矩形组成的建筑物或构筑物的场地平整工程量计算。二、基础土方开挖计算开挖土方计算规则（1）、清单规则：挖基础土方按设计图示尺寸以基础垫层底面积乘挖土深度计算。（2）、定额规则：人工或机械挖土方的体积应按槽底面积乘以挖土深度计算。槽底面积应以槽底的长乘以槽底的宽，槽底长和宽是指基础底宽外加工作面，当需要放坡时，应将放坡的土方量合并于总土方量中。 2、开挖土方计算公式：（1）、清单计算挖土方的体积：土方体积＝挖土方的底面积×挖土深度。（2）、定额规则：基槽开挖：V=（A+2C+K×H）H×L。式中：V———基槽土方量；A———槽底宽度；C———工作面宽度；H———基槽深度；L———基槽长度。. 其中外墙基槽长度以外墙中心线计算，内墙基槽长度以内墙净长计算，交接重合出不予扣除。基坑开挖：V=1/6H[A×B+a×b+(A+a)×(B+b)+a×b]。式中：V———基坑体积；A—基坑上口长度；B———基坑上口宽度；a———基坑底面长度；b———基坑底面宽度。三、回填土工程量计算规则及公式 1、基槽、基坑回填土体积=基槽（坑）挖土体积-设计室外地坪以下建（构）筑物被埋置部分的体积。式中室外地坪以下建（构）筑物被埋置部分的体积一般包括垫层、墙基础、柱基础、以及地下建筑物、构筑物等所占体积 2、室内回填土体积=主墙间净面积×回填土厚度-各种沟道所占体积主墙间净面积=S底-（L中×墙厚+L内×墙厚）式中：底———底层建筑面积；L中———外墙中心线长度；L内———内墙净长线长度。回填土厚度指室内外高差减去地面垫层、找平层、面层的总厚度，如右图：四、运土方计算规则及公式：运土是指把开挖后的多余土运至指定地点，或是在回填土不足时从指定地点取

2008年辽宁省建设工程费用标准

2008年辽宁省建设工程费用标准总说明一、辽宁省建设工程计价依据《辽宁省建设工程费用标准》(以下简称费用标准)是工程量清单计价、编制招标控制价或拦标价的依据；是编审建设工程投资估算、设计概算、施工图预算、竣工结算的依据；是调解处理工程造价纠纷、鉴定工程造价的依据；是投标报价和衡量投标报价合理性的基础；是编制投资估算指标、概算(定额)指标的基础。二、本费用标准与2008年辽宁省建设工程计价依据《A建筑工程计价定额》、《B装饰装修工程计价定额》、《C安装工程计价定额》、《D市政工程计价定额》、《E园林绿化工程计定额》配套使用。三、本费用标准中的企业管理费、安全文明施工措施费，投标人在投标报价时，可根据本企业管理水平和工程实际适当调整，但不得低于本费用标准的90%。四、本费用标准中的规费，按核定的施工企业规费计取标准执行。五、工程量清单计价中的综合单价，应按本费用标准的“工程费用取费程序表”中的管理费和利润计算方法并考虑风险组成综合单价。六、使用1996年《全国统一房屋修缮工程预算定额辽宁省单位估价表》、1993年《全国统一市政工程预算定额辽宁省单位估价表（市政维修）》、1988年《全国统一仿古建筑预算定额》的工程，可按2008年相应计价定额的人工、材料、机械单价进行换算，按照本费用标准相应标准计取各项费用。七、2008年辽宁省建设工程计价依据计价定额材料费中不包括材料检验试验费。招投标工程由投标人在投标报价时自行确定；非招标工程，工程结算时按实际发生计入其他项目费。八、本费用标准中“×××以上”，不包括其本身，“×××以下”，包括其本身。第一部分建设工程费用组成根据建设部、财政部建标[2003]206号文件《关于印发<建筑安装工程费用项目组成>的通知》精神，结合我省实际情况，确定建设工程费用项目组成如下: 建设工程费用由直接费、间接费、利润和税金组成。一、直接费直接费由计价定额分部分项工程费和措施项目费组成。（一）、计价定额分部分项工程费：由直接工程费和技术措施费组成。直接工程费：是指施工过程中耗费的构成工程实体的各项费用。包括人工费、材料费、施工机械使用费。 1、人工费：是指直接从事建筑安装工程施工的生产工人开支的各项费用，内容包括：（1）基本工资：是指发放给生产工人的基本工资。

常石磊—北京祝福你—歌词

北京祝福你 2019-01-24 （宋祖英）爱像地球仪转来转去（刘德华）北京到世界多少里伸手可及（黄晓明）爱划过轨迹经纬两极（谭晶）万里长城从东到西烽火散去（李冰冰）北京欢迎你欢迎你（范冰冰）给世界无与伦比（韩庚）北京祝福你祝福你（孙楠）激励每一天传奇（成龙）北京祝福你（陈奕迅）城墙上聊同一个话题（韩红）祈愿飘扬旌旗（汪峰）绽放我和你（周华健）北京祝福你（李玟）星空下挂满四海霞衣（常石磊）灿烂无边无际（任贤齐）共饮天和地

（黄圣依）爱像地球仪转来转去（周笔畅）北京到世界多少里（梁咏琪）伸手可及（孙悦）爱划过轨迹（庾澄庆）经纬两极（何润东）万里长城从东到西（张梓林）烽火散去（刘亦菲）北京欢迎你（叶倩文和林子祥）欢迎你（黄奕）给世界无与伦比（胡彦斌）北京祝福你祝福你（陈楚生）激励每一天传奇（许茹芸）北京祝福你（张杰）城墙上聊同一个话题（聂玫）祈愿飘扬旌旗（李玉刚）绽放我和你（张檬和赵薇）北京祝福你 (许志安）星空下挂满四海霞衣（沙宝亮）灿烂无边无际（苏有朋）共饮天和地爱像地球仪转来转去（安以轩）北京到世界多少里伸手可及（蔡卓妍）爱划过轨迹经纬两极

(古巨基 )万里长城从东到西（容祖儿）烽火散去（周涛董卿朱军撒贝宁 )北京欢迎你欢迎你（李佳明春妮）给世界无与伦比（栗坤王业涂经纬郭玮）北京祝福你祝福你（任鲁豫朱丹陈旻警察孟昆玉）激励每一天传奇（李永波李根）北京祝福你（叶一茜田亮）城墙上聊同一个话题（郎平杨扬李小双李小鹏）祈愿飘扬旌旗（鲍春来齐秦）绽放我和你（凤凰传奇）北京祝福你 (阿鲁阿卓降央卓玛）星空下挂满四海霞衣（阿尔法克尔曼乐团开克尔曼尼斯琴格日乐）灿烂无边无际（玖月奇迹）共饮天和地（章子怡）爱像地球仪转来转去（杨幂）北京到世界多少里（莫文蔚）伸手可及（李宇春）爱划过轨迹经纬两极（郭富城）万里长城从东到西（房祖名）烽火散去

大众帕萨特领驭2.0

大众帕萨特领驭2.0介绍

车前方：采用引领潮流的宽体低俯整车格局，使车头看上去动感十足，该车的前部极具视觉冲击力，巧妙运用的横向水平线条特征，使得该车前脸具有明显的横向扩展效果，重手勾勒，气势如虹。完美的“U”型线条由发动机盖延伸至上格栅，这是大众品牌的传统特征。巨大的横向延伸的上进气格栅直接贯通整个前脸，并与两侧的头灯相衔接，毫无拖沓，浑然一体，简洁而不乏气度，动感中更添峻朗。通过立体化精致雕琢的大众徽标，彰显出高级车对于细节的考究。发动机：发动机室配备装配合理，防水措施良好，线束排列整齐，空间紧凑，整齐的布局给人一种很整齐大方的感觉，杂而不乱。它具有三项独特的先进技术：每缸５气门技术，３进２排；可变长度进气管技术，通过阀门改变进气路径长短从而提升高速功率和低速扭矩；可变凸轮轴技术，在高转速工况下获得尽量高的功率，在低转速工况下获得尽量大的扭矩。最高车速192km/h，最大功率85/5400，最大扭矩172/3500，百米加速12.2S，百公里油耗7.0L，发动机电子防盗。

车侧身：平滑、自然而又充满动感，内镶镀铬外啦式门把，与侧面水平线条浑然融为一体，优雅有型。萨特车身防擦条可以减少因为意外刮伤而造成高昂的维修费用帕萨特还配备了外后视镜电动可折叠，方便您倒车或过小道等驾驶，油箱容积62L，前悬挂：多连杆式独立悬挂，后悬挂：纵臂扭转梁式半独立悬挂。副驾驶室：该车在车内空间有许多的储物空间，是您平时放些东西非常的便利，而且他的为外形还十分的美观。室内还有很多人性化的设计，比如前后的阅读灯。后排座椅：智慧性的后排阅读灯，以及熄火延迟关断的人性化设计，年轻时尚的纵向分割，使得这款车更上一层楼。后备箱：该款帕萨特后备箱容积475L，为您的居家旅行增添了很多的便利，您可以带上多种多样的旅游用品如折叠自行车、烧烤架等等可以让你旅游更加的舒适，惬意。

《建设工程工程量清单计价规范》GB50500-2008讲解

《建设工程工程量清单计价规范》GB50500-2008讲解背景提示： 2008年7月9日，建设部发布了《建设工程工程量清单计价规范》GB50500-2008（以下简称08规范），并将在2008年12月1日施行。08规范在03规范做了补充和完善，不仅较好地解决了清单计价从2003年执行以来存在的主要问题，而且对清单计价的指导思想进行了进一步的深化，在“政府宏观调控、企业自主报价、市场形成价格”的基础上提出了“加强市场监管”的思路，以进一步强化清单计价的执行。上期我们从总体上谈了GB50500-2008的主要变化（详见《数字造价》2008年第六期热点话题栏目），为了便于造价朋友深入地了解08规范，能更快地应用08规范进行计价工作，本期我们将详细介绍08规范在计价方面的变化，包括编制工程量清单，编制投标报价，编制招标控制价。一、工程量清单的组成 08规范： 3.1.4 工程量清单应由分部分项工程量清单、措施项目清单、其他项目清单、规费项目清单、税金项目清单组成。 03规范： 3.1.3 工程量清单应由分部分项工程量清单、措施项目清单、其他项目清单。主要区别：编制工程量清单时，需要提供规费项目清单、税金项目清单。二、工程量清单的要求

08规范：3.1.2 采用工程量清单方式招标，工程量清单必须作为招标文件的组成部分，其准确性和完整性由招标人负责。 03规范：3.1.2 采用工程量清单方式招标，工程量清单必须作为招标文件的组成部分。主要区别： 1、强调招标人需要对工程量清单的准确性和完整性负责，不得利用业主身份把这部分责任转嫁给投标人； 2、上升为强制性条款。三、分部分项工程量清单内容 08规范：3.2.1 分部分项工程量清单应包括项目编码、项目名称、项目特征、计量单位和工程量。 03规范：3.2.1 分部分项工程量清单应包括项目编码、项目名称、项目特征、计量单位和工程量。主要区别： 1、在原有四统一的基础上增加项目特征，变为现在的五个要件； 2、上升为强制性条款。四、分部分项工程量清单项目编码 08规范：3.2.3 分部分项工程量清单的项目编码，应采用十二位阿拉伯数字表示。一至九位应按附录的规定设置，十至十二位应根据拟建工程的工程量清单项目名称设置，同一招标工程的项目编码不得有重码。

北京祝福你和北京欢迎你歌词

北京祝福你爱像地球仪转来转去（宋祖英）北京到世界多少里伸手可及（刘德华）爱划过轨迹经纬两极（黄晓明）万里长城从东到西烽火散去（谭晶）北京欢迎你欢迎你（李冰冰）给世界无与伦比（范冰冰）北京祝福你祝福你（韩庚）激励每一天传奇（孙楠）北京祝福你（成龙）城墙上聊同一个话题（陈奕迅）祈愿飘扬旌旗（韩红）绽放我和你（汪峰）北京祝福你（周华健）星空下挂满四海霞衣（李玟）灿烂无边无际（常石磊）共饮天和地（任贤齐）爱像地球仪转来转去（黄圣依）北京到世界多少里（周笔畅）伸手可及（梁咏琪）爱划过轨迹（孙悦）经纬两极（庾澄庆）万里长城从东到西（何润东）烽火散去（张梓林）弹钢琴（李云迪）北京欢迎你（刘亦菲）欢迎你（叶倩文和林子祥）给世界无与伦比（黄奕）北京祝福你祝福你（胡彦斌）激励每一天传奇（陈楚生）北京祝福你（许茹芸）城墙上聊同一个话题（张杰）祈愿飘扬旌旗（聂玫）绽放我和你（李玉刚）北京祝福你（张檬和赵薇）星空下挂满四海霞衣(许志安）灿烂无边无际（沙宝亮）共饮天和地（苏有朋）爱像地球仪转来转去（徐若瑄）北京到世界多少里伸手可及（安以轩）爱划过轨迹(黄征) 经纬两极（蔡卓妍）万里长城从东到西(古巨基) 烽火散去（容祖儿）北京欢迎你欢迎你（周涛董卿朱军撒贝宁) 给世界无与伦比（李佳明春妮）北京祝福你祝福你（栗坤王业涂经纬郭玮）激励每一天传奇（任鲁豫朱丹陈旻）警察（孟昆玉）

北京祝福你（李永波李根）城墙上聊同一个话题（叶一茜田亮）祈愿飘扬旌旗（郎平杨扬李小双李小鹏）绽放我和你（鲍春来齐秦）北京祝福你（凤凰传奇）星空下挂满四海霞衣(阿鲁阿卓降央卓玛）灿烂无边无际（阿尔法克尔曼乐团开克尔曼尼斯琴格日乐）共饮天和地（玖月奇迹）爱像地球仪转来转去（章子怡）北京到世界多少里（杨幂）伸手可及（莫文蔚）爱划过轨迹经纬两极（李宇春）万里长城从东到西（郭富城）烽火散去（房祖名）北京欢迎你（王学圻）欢迎你（陈慧琳）给世界无与伦比（张信哲）北京祝福你祝福你（谭咏麟&李克勤）激励每一天传奇（吴辰君&陈小朵）北京祝福你（李炜&庞龙）城墙上聊同一个话题（黄品源&苗圃）祈愿飘扬旌旗（景甜&白雪）绽放我和你（萨顶顶）北京祝福你（刘涛）星空下挂满四海霞衣(胡维纳&林志玲）灿烂无边无际（刘岩&胡夏）共饮天和地（李健黄嘉果）北京祝福你（阎维文）城墙上聊同一个话题（李思思）祈愿飘扬旌旗（雷佳）绽放我和你（王宏伟&杨洪基）北京祝福你（蔡国庆&刘一祯）星空下挂满四海霞衣(吕薇李丹阳）灿烂无边无际（王丽达%刘媛媛）共饮天和地（宋祖英）合：按照屏幕顺序北京祝福你（谭晶姚星彤）城墙上聊同一个话题（林鹏陈奕迅）祈愿飘扬旌旗（黄小琥陈小朵）绽放我和你（李宇春孙楠）北京祝福你（李娜周艳泓）星空下挂满四海霞衣（辛晓琪胡彦斌张杰）灿烂无边无际（至上励合海鸣威）共饮天和地（刘乃奇田彦李振涛V4女子乐团）共-饮-天和地（常石磊任贤齐黄晓明范冰冰刘亦菲李冰冰徐若瑄韩庚张蒙房祖名张信哲汪峰董卿萨顶顶成龙安以轩）祝福你（凡子零零组合姜佳祉） Bless you 祝福你(何超仪米晓燕爱新觉罗启星李云迪刘媛媛刘亦菲)

北京欢迎你(拼音版)

北b ěi 京j īng 欢hu ān 迎y íng 你n ǐ 迎y íng 接ji ē另l ìng 一y ī个g a晨ch ?n 曦x ī 带d ài 来l ái 全qu án 新x īn 空k ōng 气q ì----陈ch ?n 天ti ān 佳ji ā 气qì息xī改gǎi 变biàn 情qíng 味wai 不bù变biàn 茶chá香xiāng 飘piāo 满mǎn 情qíng 谊yì----刘liú欢huān 我w ǒ家ji ā大d à门m ?n 常ch áng 打d ǎ开k āi 开k āi 放f àng 怀hu ái 抱b ào 等d ěng 你n ǐ----那n à英y īng 拥y ōng 抱b ào 过gu ? 就ji ù有y ǒu 了le 默m ?契q ì 你n ǐ会hu ì爱ài 上sh àng 这zh a里l ǐ----孙s ūn 燕y àn 姿z ī 不管b ùgu ǎn 远近yu ǎnj ìn 都是d ōush ì客人k ar ?n 请q ǐng 不用b úy ?ng 客k a气qi ----孙s ūn 悦yu a 相约xi āngyu ē好h ǎo 了le 在z ài 一起y ìq ǐ 我们w ǒmen 欢迎hu āny íng 你n ǐ----王w áng 力l ì宏h ?ng 我w ǒ家种ji āzh ?ng 着zhe 万年青w ànni ánq īng 开放k āif àng 每m ěi 段du àn 传奇chu ánq í----韩h án 红h ?ng 为w ?i 传chu án 统t ǒng 的de 土t ǔ壤r ǎng 播b ō种zh ǒng 为w ?i 你n ǐ留li ú下xi à回hu í忆y ì----周zh ōu 华hu á健ji àn 陌生m ?sh ēng 熟悉sh úx ī 都是d ōush ì客人k ar ?n 请q ǐng 不用b úy ?ng 客气k aqi ----梁li áng 咏y ǒng 琪q í 第几d ìj ǐ次c ì来l ái 没关系m ?igu ānxi 有y ǒu 太t ài 多du ō话题hu àt í----羽y ǔ泉qu án 北京b ěij īng 欢迎hu āny íng 你n ǐ 为w ?i 你n ǐ开天辟地k āiti ānp ìd ì ----全体qu ánt ǐ群q ún 唱ch àng 流动li úd ?ng 中zh ōng 的de 魅力m ail ì 充满ch ōngm ǎn 着zhe 朝气zh āoq ì----任r an 贤xi án 齐q í 北京b ěij īng 欢迎hu āny íng 你n ǐ 在z ài 太阳t àiy áng 下xi à分享f ēnxi ǎng 呼吸h ūx ī----蔡依林c àiy īl ín 在z ài 黄土地hu ángt ǔd ì 刷新shu āx īn 成ch ?ng 绩j ì----孙s ūn 悦yu a 我w ǒ家ji ā大门d àm ?n 常ch áng 打开d ǎk āi 开怀k āihu ái 容纳r ?ngn à天地ti ānd ì----周zh ōu 笔b ǐ畅ch àng 岁月su ìyu a绽放zh ànf àng 青春q īngch ūn 笑容xi àor ?ng 迎接y íngji ē这个zh age 日期r ìq ī----韦w ?i 唯w ?i 天ti ān 大d à地d ì大d à 都d ōu 是sh ì朋友p ?ngy ǒu 请q ǐng 不用b úy ?ng 客气k aqi ----黄hu áng 晓xi ǎo 明m íng

2008清单计算规则.

关于执行《建设工程工程量清单计价规范》(GB50500-2008)若干意见的通知

关于执行《建设工程工程量清单计价规范》（GB50500-2008）若干意见的通知日期： 2009年4月 30日【文字大小：大中小】【打印】【关闭】通知京造定〔2009〕7号各有关单位：为了促进建筑市场各方主体贯彻执行国家标准《建设工程工程量清单计价规范》（GB50500-2008）（以下简称计价规范），规范我市建设工程工程量清单计价行为，根据《关于贯彻实施<建设工程工程量清单计价规范>（GB50500-2008）的通知》（京建市〔2009〕24号）。现结合我市工程造价管理的有关规定及2001年《北京市建设工程预算定额》（以下简称预算定额），将执行计价规范中若干意见的通知发给你们，请遵照执行。一、规费发包方在编制招标文件以及发承包双方签订施工合同时，不得将规费作为竞争性费用。招标人在编制招标控制价时，应按照“关于调整2001年《北京市建设工程预算定额》规费计算方法的有关规定”（京造定〔2009〕6号）中的相关规定计算。

投标人在投标报价时，应根据有关文件规定结合本企业缴费情况自行确定。二、漏项及新增项目综合单价确定的原则漏项是指在合同承包范围内的工作内容，且计价规范中有单独列项的要求，或按计价规范要求应当单独列项，但工程量清单中没有单独列项的项目。对于漏项及新增项目等需要重新确定的综合单价（合同中没有适用或类似的综合单价），发、承包双方必须在合同中对以下内容进行约定：消耗量确定的依据；人工、材料、机械单价的确定原则；综合单价中管理费、利润、风险费的取费标准等作出明确约定。若合同中未作约定时，漏项或新增项目综合单价按以下原则确定： 1.消耗量：可依据现行定额相关项目及有关规定的消耗量确定。 2.人工、材料、机械价格：按发、承包双方确认的市场价格或参照施工期《北京工程造价信息》中的价格确定。 3.综合单价中各项取费标准：按相应项目原投标费率确定。三、综合单价中一定范围内风险及幅度的约定 1.风险范围和幅度的确定。采用工程量清单计价的工程，应在招标文件或合同中明确风险内容及其范围、幅度，不得采用无限风险、所有风险或类似语句规定风险范围及幅度。主要材料以及人工和机械风险幅度在±3%～±6%区间内考虑。 2.风险幅度变化确定原则。变化幅度应以《北京工程造价信息》中的市场信息价格（以下简称造价信息价格）为依据，造价信息价格中有上、下限的，以下限为准，造价信息价格中没有的，按发、承包人共同确认的市场价格为准。施工期市场价格以发、承包人共同确认的价格（以下简称确认价格）为准。若发、承包人未能就共同确认价格达成一致，可以参考造价信息价格。

北京欢迎你主持人串词;北京欢迎你歌词

北京欢迎你主持人串词；北京欢迎你歌词北京欢迎你主持人串词；有一种歌曲历经岁月沧桑的洗涤，当我们再次唱起，依旧澎湃着激情。有一种难忘的旋律鼓舞我们前进的斗志；让我们满怀信心，奔向崭新的黎明。下面请欣赏由XXX带来的歌曲《北京欢迎你》北京欢迎你歌词；迎接另一个晨曦，带来全新空气（陈天佳）气息改变情味不变，茶香飘满情谊（刘欢）我家大门常打开，开放怀抱等你（那英）拥抱过就有了默契，你会爱上这里（孙燕姿）不管远近都是客人请不用客气（孙悦）相约好了在一起，我们欢迎你（孙悦）我家种着万年青，开放每段传奇（韩红）为传统的土壤播种，为你留下回忆（周华健）陌生熟悉都是客人请不用拘礼（梁咏琪）第几次来没关系，有太多话题（羽泉）北京欢迎你，为你开天辟地（成龙）流动中的魅力充满着朝气（任贤齐）

北京欢迎你，在太阳下分享呼吸（蔡依林）在黄土地刷新成绩（孙楠）我家大门常打开，开怀容纳天地（周笔畅）岁月绽放青春笑容，迎接这个日期（韦唯）天大地大都是朋友请不用客气（黄晓明）画意诗情带笑意，只为等待你（韩庚）北京欢迎你像音乐感动你（汪峰）让我们都加油去超越自己(莫文蔚) 北京欢迎你，有梦想谁都了不起（谭晶）有勇气就会有奇迹（陈奕迅）北京欢迎你，为你开天辟地（阎维文）流动中的魅力充满着朝气（戴玉强）北京欢迎你，在太阳下分享呼吸（王霞李双松）在黄土地刷新成绩（廖昌永）北京欢迎你，像音乐感动你（林依轮）让我们都加油去超越自己（张娜拉）北京欢迎你，有梦想谁都了不起（林俊杰）

有勇气就会有奇迹（阿杜）我家大门常打开开放怀抱等你（容祖儿）拥抱过就有了默契，你会爱上这里（李宇春）不管远近都是客人请不用客气（黄大炜）相约好了在一起，我们欢迎你（陈坤）北京欢迎你为你开天辟地（谢霆锋）流动中的魅力充满着朝气（韩磊）北京欢迎你，在太阳下分享呼吸（徐若瑄）在黄土地刷新成绩（费翔）我家大门常打开开怀容纳天地（汤灿）岁月绽放青春笑容迎接这个日期（林志玲张梓琳）天大地大都是朋友请不用客气（张靓颖）画意诗情带笑意，只为等待你许茹芸（伍思凯）北京欢迎你，像音乐感动你（杨坤范玮琪）让我们都加油去超越自己游鸿明（周晓欧）北京欢迎你，有梦想谁都了不起（沙宝亮满文军）有勇气就会有奇迹金海心（何润东）