Adipocyte derived hormones gene expression, resistin and visfatin, in AGS gastric cancer cell line

Adipose tissue has characteristics of an endocrine organ which releases a number of adipocyte-specific factors, known as adipocytokines. It has recently suggested that adipocytokines might play a role in pathogenesis and progression of certain cancers, especially in gastric cancer. This study has managed to investigate endogenous and/or exogenous expression of Visfatin and Resistin in gastric cancer cell line. Cell culture and semi-quantitative reverse transcription polymerase chain reaction has performed to measure mRNA and protein expression of Resistin and Visfatin in gastric cancer cell lines. ELISA test has performed for cell lysate and supernatant of cell culture to measure Resistin and Visfatin protein expression and secretion. Human gastric cancer cell line [AGS cell line] has found to express Visfatin mRNA and protein but Resistin mRNA and protein has not expressed. Visfatin has expressed endogenously in AGS human gastric cancer cells. Conversely Resistin has no expression. The results of this study has suggested that expression of adipocytokine proteins in real samples, could be a biomarker for gastric cancer

Development of a probe consist of three cosmids to enumerate the chromosome 13 on uncultured lymphocytes or amniocytes using interphase FISH

To produce a reliable probe suitable for aneuploidy detection of chromosome13 on uncultured lymphocytes and amniocytes by fluorescence in situ hybridization [FISH], we used a contig of three overlapping cosmids mapped to 13q12.3. The cosmid DNA carrying the expected sequences of human chromosome 13 was isolated from host cells and labelled with biotin-11-dUTP. The hybridization and detection conditions with FITC-Avidin were optimised using a series of cultured and uncultured lymphocytes and amniocytes. Intensive signals were detected when a combination of three overlapping cosmids was used to enumerate the chromosome 13 on interphase nuclei. An average of 87 and 85.5 percent of interphase cells prepared from lymphocytes and amniocytes showed accurate number of specific signals for chromosome 13. The results obtained in present study indicate that the probe was capable of detecting the copy number of chromosome 13 on interphase cells prepared from peripheral blood or amniotic fluid cells providing that the uncultured amniotic fluid cells are free of cytoplasmic residues, RNA and protein debris

Use of Real-Time PCR method on the amniocyte samples for prenatal diagnosis of Down syndrome

In this study, the possibility of prenatal diagnosis of Down syndrome with Real-Time PCR method was evaluated. In this context, optimization of a suitable method for purification of high quality DNA from amniotic fluid samples was also considered. Pregnant women who had the high risk of having babies with Down syndrome were selected according to the biochemical and sonographic data and referred to the amniocentesis center. The DNA of total 59 amniotic fluid samples were extracted with different methods including boiling method, salting out method, Procedures of DNA extraction from Blood and Cell Culture by DNP_ Kit [CitmaGen], Procedure of DNA extraction from cells by DNA Isolation Kit for cells and tissues [Roche], Procedure of DNA extraction from Tissue by MagNa Pure DNA Isolation kit [Roche], and QIAamp DNA Micro Kit [Qiagen]. Then, the quality and quantity of the extracted DNA were evaluated by the NanoDrop ND- 1000 spectrophotometer device. Real-Time PCR reaction using fluorescent dye SYBR Green I [Applied Biosystems, UK] was performed to specifically amplify DSCAM and DYRK1A2 genes and the reference gene [PMP22]. Data analysis was performed using comparative cycle threshold method for the determination of the gene dosage and determining the number of copies of chromosome 21. This study showed that DNA extracted from amniotic fluid samples using QIAamp DNA Micro Kit [Qiagen] has the desirable quantity and quality for Real-Time PCR. Specific proliferation of targets and reference genes was achieved and difference between normal and affected groups based on differences between their gene dosages was determined. Prenatal diagnosis of Down syndrome is feasible by the Real-Time PCR method using DNA samples from amniotic fluid cells extracted by QIAamp DNA Micro Kit [Qiagen]. The results are comparable to the corresponding results from conventional cytogenetic methods