Biomedical subjects

Wen-Li Ma

Publications and source records attributed to Wen-Li Ma.

34 records · Page 2Linked to original sources

PubMed2002

A method for evaluation of the quality of DNA microarray spots.

To establish a method to evaluate the quality of the printed microarray and DNA fragments' immobilization. The target gene fragments that were made with the restriction display PCR (RD-PCR) technique were printed on a superamine modified glass slide, then immobilized with UV cross-linking and heat. This chip was hybridized with universal primers that were labeled with cy3-dUTP, as well as cDNA that was labeled with cy3-dCTP, as the conventional protocol. Most of the target gene fragments on the chip showed positive signals, but the negative control showed no signal, and vice versa. We established a method that enables an effective evaluation of the quality of the microarrays.

Nucleic Acid Hybridization↗

PubMed2002

Application of restriction display PCR in preparing the probes of the gene chip for investigating gene expression profile of K562 cells.

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.

DNA Probes↗

PubMed2002

[Purification of PCR products with cetyltrimethylammonium bromide].

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.

Cetrimonium↗

PubMed2002

[Preliminary study of development of gene chips for HIV diagnosis].

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.

Diagnostic Techniques and Procedures↗

PubMed2002

Preliminary study of DNA microarray of human placenta gene expression profile.

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.

Female↗

PubMed2002

[Cloning of an expressed sequence tag with restriction display polymerase chain reaction].

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.

Base Sequence↗

PubMed2002

[Investigation of the gene expression profile in spermatocytes].

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.

Gene Expression Profiling↗

PubMed2002

[Cloning E. coli mRNAs with poly (A) tails by restriction digest polymerase chain reaction].

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.

Base Sequence↗

PubMed2002

[Effect of probe purification on the reutilization of gene chip].

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.

DNA, Complementary↗

PubMed2002

[Detection of cell apoptosis by MTT assay].

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.

Antineoplastic Agents↗

PubMed2002

[Construction and identification of cDNA library of E.coli mRNA with poly(A) tracts].

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.

Cloning, Molecular↗

PubMed2002

[Application of Agilent 2100 Bioanalyzer in the study of differential gene expression].

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.

DNA, Complementary↗

PubMed2002

[Proliferation and denucleation of K562 cells induced by cytochalasin B].

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.

Cell Division↗

PubMed2002

[A method for construction of restriction cDNA library].

A kind of cDNA library constructed according to Restriction Display PCR(RD-PCR) technology setup in our lab,which was called restriction cDNA library,was introduced in this paper. In the construction of cDNA library, cDNA was digested with restriction endonuclease, linked with special adaptor, amplified with PCR in groups. Each group of the restriction cDNA library contained special cDNAs. The method greatly reduced the repetitive frequency and accelerated the speed of identification.

English Abstract↗

PubMed2001

Cloning and sequence analysis of HIV-1 gene fragments isolated by restriction digest polymerase chain reaction method.

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.

Journal Article↗

PubMed2000

DNA Microarray Chips Made on Surface of Ceramic Slides.

As a novel technology, DNA microarray chips are being used in gene detections. To make DNA chips capable of multiple probing, ceramic slides, instead of silicon wafer or glass slides, were used as the substrate upon which DNA microarray are deposited. Our study indicates that the ceramic DNA microarray chips can be applied to multiple hybridization and detection procedures, showing high specificity. The advantages in using ceramic substrates are discussed.

Journal Article↗