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Objective: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray., Methods: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases., Results: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes., Conclusion: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

Objective: To detect the possible presence of AI-2 quorum-sensing pathway and construct group B Streptococcus (GBS) mutants with deletion of LuxS gene related to quorum-sensing pathway., Method: V. harveyi BB170 was employed as the reporter strain to detect AI-2 pathway in GBS, and identification of LuxS homologous gene in GBS type V strain 2603 was performed by software-based analysis. LuxS gene deletion mutant Delta LusX was then constructed in GBS by means of allelic exchange and verified by Southern hybridization analysis., Results: A component in the secretions of GBS could induce bioluminescence activity in the reporter strain, suggesting the presence of AI-2 quorum-sensing pathway in GBS. luxS homologous gene was detected in GBS and LuxS gene deletion mutants was successfully constructed in GBS Ia 515 and V 2603 strains., Conclusion: This study establishes a bacterial model for studying the role of LuxS molecule in AI-2 quorum-sensing pathway in GBS and provides new insights into virulence regulation mechanism of GBS.

Objective: To establish a model for dynamic detection of adipocyte differentiation in vivo by green fluorescent protein (GFP)., Methods: The promoter of peroxisome proliferators-activated receptor gamma 2 (PPARgamma2) gene was amplified by PCR from the genomic DNA of human preadipocytes, inserted into the eukaryotic expression vector pEGFP-1 and transferred into NIH3T3 fibroblasts and preadipocytes which were induced to differentiate into adipocyte., Results: Upon induction with insulin, dexamethasone and 3-isobutyl-1-methyl xanthine, the transformed human preadipocytes differentiated into mature adipocytes and expressed GFP, but NIH3T3 fibroblasts could not differentiate into adipocytes or express GFP., Conclusion: Transformation of human preadipocytes with the recombinant of PPARgamma2 promoter with GFP gene can be employed to dynamically detect the differentiation of adipocytes in vivo.

Objective: To study two amine modification procedures for DNA microarray preparation based on polymeric coatings., Methods: One of the proposed approaches utilized poly-amine coating of silanized slides activated by 1,4-phenylene diisothiocyanate, and the other employed acrylic acid-co-acrylamide copolymer and 1-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide as the coating agents and activator, respectively. The modified slides were used for preparing lambda phage DNA microarrays, whose properties were analyzed by hybridization., Results: Formation of dendrimeric structure and polymer was observed on the surface of the slides. The signal spots in uniform, steady and regular shape, in comparison with the commercial CMT-GAPS slides, indicated successful manufacture of the microarrays., Conclusion: The two platforms are suitable for microarray preparation, and the method of acrylic acid-co-acrylamide copolymer modification is more preferred.

Objective: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells., Methods: siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting., Results: Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate., Conclusion: RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.

Objective: To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells., Methods: siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via Lipofectamine2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference., Results: Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G(1)-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%; as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels., Conclusion: RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.

Objective: To clone the obesity gene of Chinese and express human leptin in E.coli., Method: The obesity gene was amplified from the total RNA isolated from cultured human adipocytes of Chinese by reverse transcriptional PCR, inserted into TA-vector and cloned into the expression plasmid pBV220 after sequence identification., Results: DNA sequencing confirmed that the isolated obesity gene was identical to the previously reported sequence. The recombinant plasmid pBV220-OB was constructed and leptin successfully expressed in E.coli., Conclusion: Successful cloning and expression of human obesity gene in E.coli may facilitate further research of the mechanism of fat metabolism and adipocyte differentiation.

Objective: To develop a new method for fluorescent labeling technique, universal primer U(2) labeling (UPL), for microarray studies., Method: Influenza virus RNA was labeled with four labeling methods, namely UPL, random primer, restriction display incorporation labeling method and reverse transcription coupled random primer spiking labeling method (RT-PSL), respectively, and hybridized to influenza virus oligonucleotide microarray. The signals extracted from the microarrays were analyzed with SPSS 10.0 software to compare the efficiency and reproducibility of the labeling methods., Results: The fluorescence intensity, signal-to-noise ratio (SNR), true positive ratio (TPR) of the probes and reproducibility of labeling with UPL were comparable with those RD-labeling method, and higher than those of RT-PSL method. UPL reduced the complexity of the procedures in comparison with the other labeling methods., Conclusion: UPL labeling method can be used in research and development of the microarray technique.

Objective: To develop an oligonucleotide microarray for fast detection of human immunodeficiency virus (HIV)., Methods: With complete genome sequence of HIV-1 subtype B (U26942) as the target sequence and bioinformatics software such as DNAClub, Oligo6.0, BLAST, Alignment, oligonucleotide probes of high specificity with identical length and similar melting temperature (T(m)) were designed and synthesized. Oligonucleotide microarray was prepared using Cartesian Microarrayer. Using the plasmids of HIV-1 subtype B(U26942), C(U46016), F(AF075703), G(AF061640) and restriction display technique, Cy3-labeled HIV DNA fragments were amplified and the hybridization results were scanned and analyzed with Array-Pro., Results and Conclusion: Twenty-two optimized oligonucleotide microarray probes were obtained and used to prepare the oligonucleotide microarray for further screening studies. The microarray prepared significantly enhanced the sensitivity, reliability and speed of DNA assay, and possesses the potential for application in clinical setting.

Objective: To design and clone all known and predicted coding genes of Epstein-Barr virus (EBV) as the cDNA probes for preparing the microarray for EBV detection, thereby to facilitate further investigation of the pathogenetic role of EBV., Methods: Oligo 6.0 software, BLAST program and Primer Premier 5 software were employed to design and screen the cDNA probes of the whole EBV genome, whose length ranged from 300 to 600 mer each with high specificity. These cDNA probes obtained through PCR and reverse transcriptase (RT)-PCR amplification from the genomic DNA and RNA of B95-8 cells and nasopharyngeal carcinoma (NPC) tissue were cloned into T/A clone vector, followed by identification of these probes by sequencing analysis., Result and Conclusion: A total of 85 gene fragments (BWRF1 gene-contained 7 repeats of open reading frames) coding for proteins and 2 EBERs in EBV genome were successfully cloned, not including LF1 and LF3 genes that did not exist in EBV genome of B95-8 cells, which provides the basis for preparing microarray to explore the role of EBV genome in its related diseases.

Objective: To explore an effective method for detecting human papillomavirus (HPV) DNA in cervical cancer tissue., Methods: HPV L1 gene fragment in cervical cancer tissue was amplified by HPV-specific PCR with consensus primers, and typing of the HPV strains was performed on the basis of sequence analysis of the PCR product., Results: The positivity rates of HPV DNA was 78% in the 50 cases of cervical cancer, and mixed infection with HPV16 and HPV18 strains was the most common, which accounted for 48% on the total infections. Infection with HPV58 was detected in one case. The sequencing results showed no difference in L1 sequence between the detected samples and the standard German HPV58 strain., Conclusion: PCR and direct sequencing approach is effective for detecting and typing of HPV DNA in cervical cancer tissue, through which rare HPV strain or mutants of known HPV strains may not escape detection.

Objective: To modify conventional poly-L-lysine coating for oligonucleotide microarray preparation so as to enhance the sensitivity of the microarray., Method: The proposed chemical approaches included silanizing the slides with 3-glycid-oxypropyltrimethoxysilane (GOPS) after cleaning, followed by slide coating with polymers (poly-L-lysine) that was covalently bound to the modified glass. Subsequent attachment of the oligonucleotide to the modified slide surface was achieved after 1,4-phenylene diisothiocyanate (PDITC) activation of the surface. Various experiments were carried out, such as the immobilization efficiency and hybridization assays to test the modified slides, which were then used tentatively in the preparation of microarrays for SARS coronavirus detection., Results: The improved surface had high immobilization efficiency, good uniformity and satisfactory hybridization efficiency, better than those slides with conventional poly-L-lysine coating. In addition, such modified slides rendered the microarrays more resistant to consecutive probing/stripping cycles., Conclusion: The modified slide surface is satisfactory to immobilize unmodified oligonucleotide by covalent binding, which enhances not only the sensitivity of the prepared oligonucleotide microarray but also the binding of the oligonucleotide to the slide surface.

Objective: To establish a fast and simple method for screening mutant yeast strains., Materials and Methods: Homologous recombination technique was used to detect mutant yeast strains in yeast genomic library, with the green fluorescent protein gene as the reporter gene in the transposon., Results: The strains that emitted green fluorescence were isolated, indicating that the gfp gene was inserted into the yeast genome by homologous recombination., Conclusion: This study established a useful method for functional genome study by homologous recombination technique, and provide an alternative for gene therapeutic drug development.

Objective: To collect the normal spermatozoal gene expression sequence tags with the restriction display technique for constructing a microarray to understand spermatozoal gene expression profiles., Methods: The total RNA extracted from normal human spermatozoa were reversely transcribed into cDNAs, which were digested by Sau3AI and linked to universal adapters (adapter 1) at both ends. According to the sequence of the adapter, a pair of primers (universal primers 1) was designed, followed by PCR with primers 1 and the PCR products were transferred into E.coli. The positive clones were collected after identification to serve as the probes for constructing the gene expression microarray of spermatozoa by printing those probes on the slides. The accomplished microarrays were examined by Cy3-labeled normal spermatozoal samples., Results: Altogether 1 859 probes were collected, from which 368 were picked out randomly for constructing the microarray., Conclusions: Human spermatozoa contain a rich repertoire of RNAs, and the probes we prepared possess good incredibility and speciality.

Objective: To study the method for using a 70-mer oligo microarray as the probe to isolate target genes from the cDNA restriction fragments., Method: Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock culture and reversely transcribed into the double-stranded cDNAs, which were prepared into restriction cDNA fragments using restriction display (RD) method. The microarray was printed using a single 70-mer specific oligo designed to according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled by PCR method with the Cy5 universal primer before hybridization with the microarray. The microarray was stripped after washing and scanning, and the strip solution was collected for another round of PCR amplification using the universal primer without fluorescence. The PCR product was then cloned into PUC18 T vector and transformed into to E.coli JM109 cells for amplification, and the plasmids were extracted and sequenced for identification., Results: BLAST results showed that the target gene was cloned successfully., Conclusion: The target gene can be isolated directly using the 70-mer oligo microarray as the probe from the cDNA fragments prepared by RD method, without the necessity of building a cDNA library. This method can also be used in further research to acquire the differentially expressed genes after the oligo microarray hybridization.

Objective: To investigate the differences in serum biochemistry between specific pathogen-free (SPF) and conventional aged Wistar rats., Methods: Coulter-JT Analyzer was used to measure the values of serum biochemistry in the two grades of rats., Results: The serum levels of alanine aminotransferase (ALT), total protein (TP), alkaline phosphatase (ALP), total cholesterol (TC), triglyceride (TG), blood urea nitrogen (BUN), creatinine, Fe, P, glucose, uric acid (UA), and low density lipoprotein (LDL) were very significantly different between male and female Wistar rats of either conventional or SPF grade (P<0.01), which also had significant difference in albumin, lactate dehydrogenase (LDH) and apolipoprotein B (ApoB) (P<0.05). Between male aged Wistar rats of the two grades, the differences of TP, albumin, albumin/globulin (A/G) ratio, TC, TG, blood glucose, ApoA1, ApoB, UA, high-density lipoprotein (HDL), LDL, and glutamic oxalacetic transaminase (GOT) were very significant (P<0.01), with also significant differences in ALT, Fe, Mg (P<0.05). Between the female rats of the two grades, the serum levels of ALT, TP, albumin, A/G ratio, ALP, TG, BUN, creatinine, Fe, ApoA1, APOB, HDL, LDL, and bile acids were very significantly different (P<0.01), and Mg was significantly different (P<0.05)., Conclusion: Different microbiological profiles affect serum biochemistry of aged Wistar rats.

Objective: To screen tumor-specific genes of K562 cells using DNA microarray technique., Methods: The genomic DNA of normal white blood cells and cultured K562 cells were respectively purified and digested with Sau3A I, and the digested DNA fragments of K562 cells were cloned into TA cloning vector to construct the corresponding genomic DNA library. The insert genomic DNA fragments were amplified from the library to prepare the microarray using Cartesian 5500 Microarrayer. The digested genomic DNA fragments of normal white blood cells were labeled with fluorescent Cy3 by restriction display PCR (RD-PCR), followed by hybridization with the microarray, after which the slide was washed and scanned with ScanArray., Results: Among the 426 target genes, 42 differential genes were identified in the genomic DNA of K562 cells in comparison with the normal white blood cells. One of the genes was identified as the breakpoint cluster gene (BCR) after sequence analysis., Conclusions: The DNA microarrays we constructed may effectively identify the tumor-specific genes in K562 cells, and DNA microarray technique can be helpful in elucidating the molecular mechanisms of tumorigenesis at the genomic level.

Objective: To investigate the expression of cyclin-dependent kinase-2 (CDK-2) gene in SH-SY5Y cells., Methods: The expression of CDK-2 gene was examined with reverse transcriptional (RT)-PCR, and the PCR products underwent electrophoresis on non-denaturing poly-acrylamide gel (PAG) followed by silver staining. The separated and purified DNAs were ligated into pMD18-T vector, and the positive clones identified by sequence analysis., Results: Two DNA bands were displayed on PAG, and the one with smaller molecular weight was less intensively stained. The sequences of the two clones indicated that both were products of CDK-2 gene., Conclusion: Two kinds of CDK-2 gene products are co-expressed in the SH-SY5Y cells, one of which lacks the fifth exon and is expressed at a low level.

Objective: To study the preparation of oligonucleotide microarray for detecting vaccinia virus., Methods: Oligonucleotide probes were designed and synthesized according to the specific genes of vaccinia virus. Sample DNA of the virus and the negative control sample were obtained and labeled by restriction display technique, followed by hybridization to the oligonucleotide microarray and scanned by Agilent scanner., Results: Strong hybridization signals were detected from the viral DNA hybridized with the microarray, but were absent in the negative sample when positive probes were not used., Conclusion: Distinct differences in the hybridization signals between the virus sample and negative sample and between the samples obtained in different phase of infection demonstrate high specificity and sensitivity of the microarray for vaccinia virus detection.

Objective: To prepare the microarray for detection of hepatitis D virus (HDV)., Method: Several pairs of specific PCR primers were designed according to the conserved region of HDV genome. The DNA microarray were prepared by blotting the PCR products onto the surface of glass slides with the use of robotics, and restriction display PCR (RD-PCR) was employed to label the samples., Result: Sequences analysis showed that the products of PCR amplification were the specific gene fragments of HDV. Hybridization signals on the gene chip demonstrated good specificity and sensitivity of the microarray for HDV detection., Conclusion: Microarray-based clinical HDV detection can be sensitive and effective.

Objective: To develop a DNA microarray technique for fast diagnosis of plague., Methods: Restriction display polymerase chain reaction (RD-PCR) and hybridization with fluorescently labeled cy5 were employed for detecting the sample DNA of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica., Results: The prepared microarray is capable of distinguishing Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica in the same genus., Conclusion: The constructed DNA microarray is an effective diagnostic tool for the detection of Yersinia pestis.

Objective: To amplify and clone the N gene of severe acute respiratory syndrome-associated coronavirus., Method: Using primer Premier 5.0 software, two pairs of nested PCR primers were designed to amplify the N gene. After purification, the amplified products were cloned into pMD18-T vectors, and the positive clones with the inserted fragments were identified by sequence analysis., Results: The amplified products was about 1 375 bp in length, and sequence analysis demonstrated that the N gene fragments had been successfully inserted into pMD18-T vectors., Conclusion: The successful amplification and cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.

Objective: To prepare DNA microarray probes for the detection of human parvovirus B19. Method Specific PCR primers were designed with the Primer Premier 5.0 to amplify the conserved regions of human parvovirus B19 genome. The PCR products were cloned into the pMD-18 T vector., Method: Sequences analysis showed the PCR products conformed to the sequences contained in the genome of human parvovirus B19., Result: Sequences analysis showed the PCR products conformed to the sequences contained in the genome of human parvovirus B19., Conclusion: PCR amplification of the conserved and specific human parvovirus B19 genes is simple and effective to prepare the desired probes.

Objective: To design oligonucleotide probes for microarray detection of dengue virus., Methods: By analyzing the cDNAs of dengue viruses of 4 different serotypes with BLAST program, a group of specific sequences for the candidate probes was acquired. Oligo6.0 software was applied to analyze the candidates to select the probes with high specificity, identical length and similar melting temperature (Tm)., Result: Altogether 48 oligonucleotide probes were designed, and deposited on oligonucleotide chips as the microarray for dengue virus detection., Conclusion: BLAST program and Oligo6.0 software are simple and effective means for designing the oligonucleotide probes.

Objective: To study the method for rapid preparation of the gene chip probes for Bacillus anthracis protective antigen (pag)., Methods: According to the sequences of pag published on PubMed, a pair of primers was designed to amplify the PA gene of Bacillus anthracis. Restriction enzyme digestion and AT clone were employed to rapidly screen out the recons of PA segments for the preparation of the gene chip probes. The DNA sequences of these segments were determined with 377 DNA Sequencer, and the resultant sequences analyzed with Bioinformatic software., Results: With the designed primers, the pag 2,205 bp in length was amplified. After enzyme digestion and AT clone, 7 fragments of varied lengths were screened, which underwent analysis with the basic local alignment sequence tool (BLAST) and 377 DNA Sequencer. BLAST analysis showed that all the fragments cloned belonged to Bacillus anthracis., Conclusion: PCR in conjunction with restriction enzyme digestion and AT clone is rapid and simple to prepare the desired gene chip probes.

Objective: To prepare DNA microarray for detecting both hepatitis B and D virus as (HBV and HDV)., Methods: With the assistance of Oligo6.4 software, specific PCR primers targeting the conserved region of HBV and HDV were designed. The PCR products were purified and cloned into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were then extracted from positive clones and the target gene fragments underwent sequence analysis., Results: The gene fragments of HBV and HDV were obtained by PCR, which were confirmed by sequence analysis to be specific gene fragments of HBV and HDV., Conclusion: Using PCR amplification products to prepare the DNA microarray is quick, simple and effective.

Objective: To investigate the expression profile of angiotensin type 1 receptor (AT1R) mRNA in fibrotic and normal liver tissues., Methods: Semi-quantitative RT-PCR was used to detect the expression of AT1R mRNA in 18 specimens of fibrotic liver tissues adjacent to liver cancers and 12 normal liver tissue specimens. Specific AT1R product and GAPDH product used as internal control were both amplified by RT-PCR, and the ratio of their integrated optical densities calculated to estimate the relative quantity of AT1R mRNA expression., Results: The relative mRNA expression of AT1R in fibrotic liver tissues was significantly higher than that of normal liver tissues (1.27+/-0.45 vs 0.71+/-0.21, P < 0.01)., Conclusion: AT1R may play an important role in the development of hepatic fibrosis.

Objective: To explore an effective means for the detection of Severe Acute Respiratory Syndrome (SARS)- associated coronavirus., Methods: The RNAs of the virus contained in the sputum samples from established SARS patients were extracted and reversely transcripted, followed by nested PCR using the reversely transcripted cDNA as the template. The PCR products were cloned then into the pMD18-T vectors, followed by sequence analysis., Results: Specific fragments were amplified from the sputum samples of SARS patients, which were confirmed by DNA cloning and sequencing to belong to SARS-associated coronavirus. The Result of Blast shows only the difference in one nucleic acid from the TOR2 strain of SARS-associated coronavirus., Conclusion: Sequence analysis has confirmed the existence of SARS-associated coronavirus in the sputum samples of SARS patients, and nested RT-PCR is a quick, easy, and convenient way for the detection of the virus.

Objective: To clone and analyze Bacillus thuringiensis gene fragments isolated by restriction digest PCR (RD-PCR)., Method: Specific primers were designed to amplify the genes of Bacillus thuringiensis israelensis (Bti), and the PCR products were classified and re-amplified by RD-PCR to obtain the fragments for subsequent purification and cloning into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced., Results: Sequence analysis showed that all the fragments amplified were Bti genes., Conclusion: RD-PCR is reliable in breaking down large gene fragments into confined and shorter gene fragments for preparing microarray probes.

Objective: To explore the feasibility of using Agilent 2100 bioanalyzer in the detection and genotyping of human papilloma virus (HPV)., Methods: The consensus sequence of highly conserved region (L1) and genotype-specific gene fragments of all HPV genotypes (including HPV6, 11, 16 and 18) were amplified by PCR technique using general and type-specific primers respectively. Agilent 2100 Bioanalyzer and routine agarose gel electrophoresis were then employed respectively to examine the PCR products, and the accuracy, reproducibility and sensitivity of these 2 detection techniques were compared., Results: Agilent 2100 Bioanalyzer showed better accuracy in determining the length of the gene fragments than agarose gel electrophoresis, the accuracy of the former reaching above 95% while the latter was only 85%. Bioanalyzer was also 100 times more sensitive than agarose electrophoresis., Conclusions: Agilent 2100 Bioanalyzer combined with PCR is specific, accurate and sensitive for HPV detection and genotyping.

Objective: To study the signal-to-noise ratio (SNR) of two restricted fluorescence labeling methods for examining gene expression profile by microarray hybridization., Method: Samples of Saccharomyces cerevisiae mRNA was labeled by traditional reverse transcription method and 2 restriction fluorescent labeling methods using respectively Cy-universal primer and extension incorporated Cy-dNTP. The labeled samples were examined by the microarray, followed by washing and scanning under the same conditions., Results: The two restriction labeling methods showed superior results with lowered background and enhanced SNR and sensitivity, and Cy-universal primer labeling presented the best results., Conclusion: SNR can be enhanced by the restriction labeling methods, which improve the applicability of microarray technology.

Objective: To observe the effect of cytochalasin B on the denucleation of K562 cells., Methods: K562 cells were grown in RPMI 1640 medium supplemented with 10% calf serum and 4 microL cytochalasin B (CB). Denucleation was induced in the cultured cells by CB, and the cells were examined by phase contrast microscopy and Giemsa staining respectively., Results: The denucleation of K562 cells induced by CB was clearly observed, and the cell proliferation was obviously inhibited., Conclusion: The denucleation efficiency of K562 cells is positively correlated with CB concentrations and the duration of CB treatment. CB at low doses may inhibit the cell proliferation and at high doses causes cell death.

Objective: To study the application of Agilent 2100 Bioanalyzer in the study of gene differential expression., Methods: The total RNAs were extracted and purified from Saccharomyces cerevisiae to synthesize double-stranded cDNAs by reverse transcription. Restriction display-PCR was employed to obtain the cDNA fragments, which were examined by Agilent 2100 Bioanalyzer and agarose gel electrophoresis., Results: The analysis showed that Agilent 2100 Bioanalyzer was more sensitive and faster to isolate and display the differentially expressed genes, providing at the same time accurate quantitative information for each fragment in the DNA sample., Conclusion: Agilent 2100 Bioanalyzer can be instrumental for the study of differential gene expression.

Objective: To construct and identify the cDNA library of E.coli mRNA with poly(A) tracts., Methods: The cDNA library of E.coli was constructed by restriction display-PCR (RD-PCR) technique, followed by sequencing and bioinformatics analyses., Results: cDNA library of E.coli mRNA with poly(A) tracts was successfully constructed, and 66 gene fragments were sequenced., Conclusion: The constructed cDNA library of E.coli mRNA with poly(A) tracts contains a low rate of repetition and is of high quality.

Objective: To investigate differential gene expression in apoptotic cells induced by As(2)O(3), and identify novel apoptosis-related genes., Method: Apoptosis of K562 cells cultured in RPMI 1640 medium supplemented with 10 % calf serum was induced by As(2)O(3). Total RNA of the apoptotic and normal cells were then extracted, purified and subject to reverse transcription into first-strand cDNA, labeled with Cy3/Cy5. Placenta DNA microarrays containing 348 DNA fragments were used to analyze the changes in gene expressions in the cells treated with As(2)O(3)., Result: Eleven differentially expressed genes were identified in the apoptotic cells in comparison with the normal cells, 3 of which were associated with apoptosis, while the others were related to cell growth and proliferation., Conclusion: The placenta DNA microarrays we constructed may well apply to the analysis of the differentially expressed genes.

Objective: To establish a method for purifying PCR products with cetyltrimethylammonium bromide (CTAB)., Method: Selective precipitation of the PCR product was performed using CTAB, which forms compound with DNA fragment in salt solution of appropriate concentration, but not with single strain oligonucleotide or dNTPs. The precipitation could be dissolved in 1.2 mol/L NaCl while the addition of ethanol caused the desired PCR product to precipitate so as to be recovered., Result: The primers and small-molecule dNTP could be effectively eliminated after this procedure. Although the output of the purification process reached only 80% that by current reagent kit, it reduces the cost to as low as 1/8 of that normally required by the kit., Conclusion: CTAB is applicable for purification of the PCR product.

Objective: To study the technology for establishing DNA chips for the diagnosis of HIV., Methods: HIV 1U26942 DNA fragments were isolated by restriction display-PCR (RD-PCR) and printed onto aminosilane-coated glass slides by Pixsys 5500 arrayer as probes to prepare the gene chips. HIV samples, after labeled with Cy3, were hybridized with the microarray followed by scanning for analysis of hybridization kinetics of the RD fragments., Results: The experimental condition for preparing the gene chips was investigated and 12 RD fragments were screened as probes for further study., Conclusion: The technique established in this study for preparing DNA chips is specific and applicable.

Objective: To collect the gene fragments of the gene chips for investigating the expression profile of K562 cells., Methods: The mRNA was extracted from cultured K562 cells to obtain cDNA by reverse transcription-PCR. The gene fragment products of different groups were acquired and isolated by reverse display PCR (RD-PCR)., Result: The probes for the gene chip were successfully prepared., Conclusion: RD-PCR adapted in this study suits the needs for collecting the probes for the gene chips.

Objective: To prepare human placenta genechip for analyzing differential gene expressions., Methods: The target gene amplified from Hybrid Hunter cDNA library was dotted onto the slides by the arrayer (PixSys 5500). Total RNA from K562 cells treated with or without arsenic trioxide was extracted, the mRNA purified and cDNA synthesized. Fluorescent labeling of the samples was performed by restriction display PCR, followed by hybridization with the microarray that was subsequently washed and scanned, with the derived signals analyzed by computer., Results: A reliable and specific method for preparing and assessing gene expression profile microarray was established, with which a total of 45 differentially expressed gene fragments were isolated., Conclusion: The genechips can be helpful in the analysis of differential gene expressions.

Objective: To isolate gene fragments from SH-SY5Y cells by way of restriction display polymerase chain reaction (RD-PCR)., Methods: Total mRNA was extracted from SH-SY5Y cells followed by synthesis of the single-strand cDNA with Oligo (dT18) as the anchored primer, and the second strand was synthesized by nick translation. The double strands were cleft with restriction enzyme Sau3A I and the fragments ligated with a universal adapter to be amplified with the universal primers and selected primers. The products were then ligated into the pMD18-T vector and sequenced., Results: One of the sequenced clones was retrieved in the National Center for Biotechnology Information (NCBI) databases with Blast program. The results showed that the sequence possessed great similarity to one fragment of the 17th chromosome in the genome. Sequence analysis with GenScan software indicated that the EST might be one section of an unknown gene., Conclusion: RD-PCR provides simple and efficient approach for isolating EST from cells, and cDNA clone sequencing combined with bioinformatics analysis may be helpful in identifying new genes.

Objective: To observe whether dendritic cells (DCs) transfected with carcinoembryonic antigen (CEA)-vaccinia recombinant virus (rV-CEA) induces cytotoxic T lymphocyte-mediated CEA-specific immunity in vitro., Methods: DCs derived from peripheral blood monocytes were transfected with rV-CEA and then cocultured with autologous T cells. The proliferation of the induced T cells and their cytotoxic activity against CEA-secreting tumor cells were assessed and compared with those of T cells induced by DCs without rV-CEA transfection., Results: T cells induced by DCs transfected with rV-CEA showed specific cytotoxicity against CEA-secreting tumor cells., Conclusion: DCs transfected with rV-CEA can elicit activation of CEA-specific T cells.

Objective: To investigate the gene expression profile in the mature spermatocytes and the molecular mechanism of the male infertility syndromes., Methods: Restriction display PCR (RD-PCR), a new method of differential display technique, was used to collect plenty of gene fragments expressed in either normal or abnormal spermatocytes, the gene expression profile of which were demonstrated by these fragments(gene expression sequence tags, ESTs), in the form of cDNA fragments length polymorphism. These ESTs will be used to make DNA microarray as probes to further explore the gene expression profile in spermatocytes and to diagnose the male infertility., Results: Lots of ESTs were expressed in either normal or abnormal spermatocytes and some differentially expressed ESTs have been collected and purified., Conclusion: Either for known or unknown genes, RD-PCR is very effective in rapid acquisition of large amount of ESTs with proper and similar length fit for making microarray as the probes.

Objective: To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli., Methods: mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters. Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors., Results: More than 100 gene fragments were cloned, 30 of which were sequenced., Conclusion: Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.

Objective: To investigate the effect of probe purity on the reutilization of gene chips., Methods: Purified and unpurified probes were respectively hybridized with the gene chip and after being scanned with Scanarry lite, a laser scanning device, the gene chip was treated with stripping solution to wash off the probes. After another round of scanning under the same condition to obtain the images, the gene chip was recycled for another hybridization., Results: Hybridization of unpurified probes resulted in high background noise and obscure positive signals, which were present even after wash with stripping solution for 4 times. Hybridization using purified probes, in contrast, presented low background noise that was completely eliminated after the gene chip was washed twice before it was used again., Conclusion: The purity of probes is of great importance to the recycling of the gene chips, and hybridization with unpurified probes renders the chips impossible for reutilization.

Objective: To study the feasibility of using MTT assay to detect cell apoptosis., Methods: K562 cells were grown in RPMI 1640 medium supplemented with 10% calf serum and 4 micromol/L arsenic trioxide. Apoptosis was induced in the cultured cells by As2O3, and the cells were detected with optical microscope, DNA gel electrophoresis and MTT staining respectively., Result: MTT staining could also accurately detect cell apoptosis, by which the apoptotic cells were easily distinguished from normal cells and dead cells., Conclusion: MTT staining is simple, convenient and practical for detecting cell apoptosis.

OBJECTIVE: To clone and analyze the HIV-1gene fragments isolated by restriction digest polymerase chain reaction (RD-PCR). METHOD: All the HIV-1 gene fragments were divided into 10 subgroups and amplified by RD-PCR. The PCR products of each subgroup were purified and cloned into the T-vectors, then identified rapidly. The plasmids were extracted from positive clones and the target gene fragments were amplified and sequenced. RESULTS: Sequences analysis showed that all the fragments amplified were HIV gene. CONCLUSION: A method for cloning and identifying multiple fragments has been improved and used for restriction fragments.