[Preparation of small interfering RNA expression cassette based on PCR technique].

OBJECTIVE: To modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference. METHODS: The U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference. RESULTS: The sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection. CONCLUSION: The siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

[Application of oligonucleotide microarray primer extension to detection of p53 single nucleotide polymorphisms].

BACKGROUND & OBJECTIVE: Increasing evidences imply that single nucleotide polymorphisms (SNPs) are involved in etiopathology, individual therapy, and prognosis of many diseases. Rapid and accurate SNPs detection in disease genes is vitally important for genomic research. To detect p53 SNPs, the method of an arrayed primer extension based on oligonucleotide microarray was developed. METHODS: Twelve extension primers were designed in exon 3 of p53 gene. These primers were synthesized and printed onto a microarray with 7x8 spots. The fragments of p53 DNA were extracted from K562 cells by nested polymerase chain reaction (PCR), and hybridized with the microarray. After hybridization, the primer extension reactions were carried out with DNA polymerase Klenow, labeled with Cy3-dUTP or Cy5-dCTP; the product was washed and scaned, and the fragments of p53 was sequenced by ABI3730 DNA Analyzer. RESULTS: The scanning result showed that the primer extension reactions were successful, and single base labeled with Cy3 or Cy5 was extended correctly at the end of 3'primers. The results of microarray SNPs detection were consistent with the results of sequencing verification, while much less complicated. CONCLUSION: These results indicate that the arrayed primer extension techniques are useful in parallel detecting SNPs of genes of interest, which is not only sensitive and accurate but also miniaturized the assays when analyzing multiple DNA targets with minimal reagents.

[Oligonucleotide microarray preparation using enhanced poly-L-lysine glass slides].

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.

[Isolation of the target gene from cDNA restriction fragments using 70-mer oligo microarray].

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.

[Analysis of gene expression patterns of leukemia K562 cells after cytochalasin B treatment].

BACKGROUND & OBJECTIVE: The advanced technique of DNA microarray makes it possible to monitor the expression of thousands of genes simultaneously in one hybridization experiment. This technique accelerates demonstration of anti-tumor drug mechanisms and discovery of new drug targets. This study was designed to investigate the differential gene expression of K562 cells after cytochalasin B treatment using cDNA microarray. METHODS: Restriction display polymerase chain reaction (RD-PCR) products of 277 human genes were spotted on a glass slide in microarray. K562 cells grew in RPMI 1640 medium with 10 microg/ml cytochalasin B. After 24 hours, the total RNA was isolated from K562 cells, and mRNA was purified. Both mRNA from the treated K562 cells and the controlled K562 cells were reversely transcribed into cDNA and labeled with two different fluorescence dyes: Cy5 or Cy3, using a method of restriction digestion and PCR labeling (RD-PCR). The probes were hybridized to the cDNA microarrays. After high-stringent washing,the cDNA microarray was scanned for the fluorescent signals and showed difference between the two cells. RESULTS: Among the 277 target genes, 18 down-regulated genes were identified after cytochalasin B treatment. CONCLUSION: There is a consistent tendency toward lower-expressed genes in partial K562 cells after cytochalasin B treatment. Most down-regulated genes were correlated with cell proliferation, signal transduction, and transcription factor.

[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.

[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.

[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.

[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.

[Analysis with DNA chips of the changes of gene expressions in K562 cells in response to As2O3 treatment].

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.

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.