ABSTRACT
Objective To evaluate the utility of fluorescent dye SYTO 13 for high -resolution melting ( HRM) detection in single nucleotide polymorphism ( SNP) genotyping and its clinical application . Methods This is a performance verification study .36 genotype defined samples were divided into three groups:SNP rs3125734 C>T (class Ⅰ SNP) ,rs255758 A>C (class ⅡSNP) and rs688C>T.These samples were used to evaluate SYTO 13′s SNP genotyping capability of class ⅠSNP, classⅡSNP, and two PCR products of different lengths (52 and 107 bp) covering the same SNP of rs688C>T.The commercial HRM dye of LCGreen Plus was used as the control .The genotyping capability is indicated by the Tm difference(ΔTm) between wild type and homozygous mutant genotypes .The Tm differences between wild genotype and homozygous mutant genotype were compared using the Independent Samples t test.Paired t test was used to evaluate genotyping capability of the two dyes .The clinical applicability is evaluated by synchronously performing PCR amplification and HRM analysis on thirty -five randomly selected DNA samples with known genotypes of the three SNPs .Results The SNPs of class Ⅰ and class Ⅱ can be genotyped directly and clearly with SYTO13 (ΔTmclas Ⅰ =0.36 ±0.05,tclas Ⅰ =14.827,Pclas Ⅰ =0.000;ΔTm clas Ⅱ =0.42 ±0.110,tclasⅡ =9.539,Pclas Ⅱ =0.000).The classⅠSNP genotyping results was better using SYTO13 (ΔTmSYTO13 =0.39 ±0.027), while the SNP genotyping for small amplicon did not discriminated clearly in this study .Long amplicons of class ⅠandⅡSNPs can be identified directly except for several samples which can be genotyped accurately after having performed reexamination .Conclusion SYTO13 can apply for HRM analysis of genotyping classⅠand ⅡSNPs with long amplicon and for clinical routine detection.
ABSTRACT
Objective To investigate the effects of c?myc promoter binding protein 1(MBP?1)gene on the proliferation of human Saos?2 osteo?sarcoma cells in vitro. Methods Saos?2 cells were divided into three groups:blank control group(untransfected cells),negative group(cells transfected with missense sequence)and experimental group(cells transfected with MBP?1 shRNA). Two MBP?1 shRNA sequences and one neg?ative control shRNA sequence were designed ,synthesized and cloned into pSIREN?retroQ plasma. Then the recombinant plasmids were construct?ed and transfected into human Saos?2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP?1 mRNA and protein in Saos?2 cells were detected by real?time PCR and Western blot ,respectively. The effects of altered expression of MBP?1 on cell proliferation were measured by CCK?8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos?2 were determined by Western blot. Results PCR and sequenc?ing results indicated that the recombinant plasmids pSIREN?retroQ was constructed. The relative expression level of MBP?1 mRNA in the MBP?1 siRNA transfection group was significantly decreased than that in blank control group(P<0.05). Compared with the blank control group,the ex?pression levels of MBP?1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos?2 cells at 48,72,and 96 hours after MBP?1 siRNA transfection were significantly increased than those in the blank control group(P<0.05). Compared with the blank con?trol group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased(P<0.05). Conclusion Knockdown of the expression of MBP?1 gene promotes the proliferation of human Saos?2 osteosarcoma cells. MBP?1 gene may become the new tar?get of gene therapy for osteosarcoma.
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Objective To silence human gene Set7/9 and screen out stable transfection cell line in hepatocellular carcinoma cell line HepG2 so as to investigate the impact of down-regulation of Set7/9 in cell line HepG2 and provide experimental foundation for studies on the effect of set7/9 in HepG2.Methods The target oligo was designed and synthesized;shRNA interference vector and the control vector were constructed and transfected into HepG2 cells;the stable transfection cells were screened out.Then Real-time PCR and Western blot were performed to detect the silence of Set7/9 according to both gene expression and protein expression level. Results The shRNA interference vector was constructed and transfected into HepG2 cells successfully.Compared with that in the negative control group,the expression of Set7/9 was dramatically downregulated (P < 0.05 ). Meanwhile,the expression of related protein Sirt1 and Suv39h1 was upregulated 8.4 folds and 1.1 fold, respectively.Conclusion Downregulation of Set7/9 expression can upregulate Sirt1 and Suv39h1,suggesting that Set7/9 may affect the activity of HepG2 cell lines.
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BACKGROUND: Talomerase activity inhibitor inhibits or kills renal carcinoma cells, and also affects stem cells that play importan roles in occurrence and development of renal carcinoma.OBJECTIVE: To observe renal carcinoma stem cell surface marker CD133 and telomerase activity expression in serum-free suspension culture, and to compare with renal carcinoma cells in serum suspension culture.DESIGN, TIME AND SETTING: The in vitro cytological study was performed at the Jiangsu University from June 2008 to Februar 2009.WIATERIALS: Fresh normal renal tissue surrounding renal carcinoma was obtained from Affiliated Hospital, Jiangsu University.Renal carcinoma stem cell line OS-RC-2 was supplied by Cell Bank, Chinese Academy of Sciences Shanghai Branch.METHODS: OS-RC-2 in logarithmic phase, digested by trypsin, and centrifuged. Supematant was removed. OS-RC-2 cell line in serum-free DMEM/F12 supplemented with epidermal growth factor and basic fibroblast growth factor was incubated at 2×105/L in 5% CO2 incubator at 37℃. Renal carcinoma cultured in serum and normal renal tissue served as controls.MAIN OUTCOME MEASURES: Cell growth was observed under an inverted microscope. Expression of CD133 and CD34 was detected using flow cytometry. Reel-time quantitative TRAP assay was applied to evaluate telomerase activity in renal carcinoma stem cells.RESULTS: After incubated in serum-free medium, renal carcinoma stem cells were round and suspended. Two days later, cell mass generated. Each cell mass contained 3-8 cells, with strong refraction. Seven days later, cell mass became more, presented big body that was regular, round or elliptical. CD133+CD34- rate in renal carcinoma stern cell mass was significantly greater in serum-free suspension culture compared with in serum suspension culture. CD133 and CD34 expression was not determined in normal renal tissue. There were significant differences among groups (F=328.25, P < 0.05). Telomerase activity was greater in renal carcinoma stem cells and renal carcinoma cells compared with normal renal ceils (F=-278.74, P < 0.05). No significant difference was detected between renal carcinoma stem cells and renal carcinoma cells.CONCLUSION: Compared with serum cultured renal carcinoma cells, serum-free cultured renal carcinoma cell surface marker CD133 presents high expression. Moreover, talomerase activity is high in renal carcinoma stem cells and renal carcinoma cells compared with normal renal tissue.