Biomedical subjects

Qiu-ye Guo

Publications and source records attributed to Qiu-ye Guo.

4 recordsLinked to original sources

PubMed2006

[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.

Gene Silencing↗

PubMed2004

[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.

Base Sequence↗

PubMed2004

[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.

DNA, Viral↗

PubMed2002

[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.

Antineoplastic Agents↗