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OBJECTIVE: To prepare the national standard substance for quantitative determination of residual DNA in mouse myeloma(NS0)cells. METHODS: NS0 cell DNA was prepared using genomic DNA purification reagents QIAGEN and Genomic-tip 500/G,analyzed for purity and concentration by UV spectrophotometry and agarose gel electrophoresis, and split in 100 microliters and frozen in screw cap tubes. Then five independent laboratories were organized to calibrate the first batch of NS0 DNA national standard using UV spectrophotometry, and evaluated the stability and applicability. RESULTS: The prepared national standard substance of NSO DNA was qualified as indicated by A260/A280 between 1.7 and 1.9 and a single specific band in agarose gel electrophoresis. The standard substance was calibrated for 90 times by the five laboratories,and the results showed a geometric mean concentration of 87.5 μg•mL-1, the 95% confidence interval of the geometric mean concentration in a single determination was 86.6-88.5 μg•mL-1. Short-term stability experiments showed that there was no significant change in the standard DNA concentration and A260/A280 ratio after being stored at 4 ℃ for 4 m. The results of storage stability test at -20 and -70 ℃ for 1 year revealed that there was no significant change in the standard DNA concentration and A260/A280 ratio, and the electrophoresis strip was single, so the standard substance was stable at -20 ℃. In applicability studies using the NS0 DNA standard substance, the real-time PCR had high sensitivity up to 0.003 pg of DNA with good linearity (r20.999) in the content range of 3 fg•μL-1-300 pg•μL-1. CONCLUSION: The prepared standard substance with batch number of 330002-201701 and DNA concentration of 87.52 μg•mL-1 is qualified in all tests and may be used as national standard substance for residual DNA assay by real-time PCR method.
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Objective Developing a rapid and accurate real-time qPCR method for the detection of HCV-RNA.Methods HCV nucleotide sequence was analysed in Clustal software and primers and probe were designed in the conserved region of 5'UTR.The reaction system optimization of real-time qPCR method was used chessboard titration,pseudoviral particles were used as quantitative standard to assess the performance.New methods was compared with clinical commonly used kit of HCV-RNA and discuss the application value.Results The sensitivity of new real-time qPCR method was 50 IU/ml,coefficient variation was less than 5％.The quantitative results of this method could be traceable to national standards of GBW09151a.40 samples were determined by new methods and clinical commonly used kit of HCV-RNA,the positive concordance rate was 100 ％,the negative concordance rate was 56 ％.14 samples were positive by new method,but negative by Qiagen kit,illustrating that the sensitivity of new method was superior to Qiagen kit.Conclusion New TaqMan-MGB probe-based real-time qPCR method is a specific,sensitive,simple,rapid and exactly used to detection of HCV-RNA.
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ABSTRACT The MYB family represents one of the most abundant classes of transcriptional regulators that perform pivotal role under different developmental processes and abiotic stresses. In present study, a MYB gene from Oryza sativa was selected for functional characterization. Bioinformatics analysis revealed that OsMYB1 cDNA encodes R2-R3 type DNA binding domain consisting of 413 amino acids having size of 44 kDa and pI of 6.24. DNA binding domain containing region was cloned and over-expressed in E. coli. Then, the survival of pGEX-OsMYB1 transformed E. coli cells was compared with control plasmid under different concentrations of NaCl, mannitol, high and low temperature. pGEX-OsMYB1 enhanced the survival of cells at high temperature and salinity. Electrophoretic mobility shift assays (EMSAs) have shown that recombinant OsMYB1 protein was able to bind with DIG labeled probe containing MYB binding site. RT-qPCR analysis revealed high MYB1 expression under wounding, salt, drought and heat stresses in rice. Expression was 23 fold higher in response to wounding demonstrating the worth of OsMYB1 up-regulation in wounding. Intrinsic disorder profile predicted that OsMYB1 exhibits 60% degree of intrinsic disorder proposing that these regions might be involved in DNA binding specificity and protein-protein interaction. The positive response of OsMYB1 suggests that its over-expression in crop plants may help in providing protection to plants to grow under abiotic stresses.
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Objective: To establish real-time qPCR method to analyze each HLA-C allele expression level of individual.Methods:Database including exon 2&3 sequences of HLA class Ⅰalleles was built ,HLA-C allelic specific primers were designed and the real-time quantitative PCR method for analyzing HLA-C allele expression level was built .The allelic specificity of these primers were confirmed in database and 835 normal peripheral blood samples of Han population .The mRNA level of each HLA-C allele from 20 pairs of liver tumor tissues and non-tumor tissues was analyzed by the qPCR method we built .Results:20 pairs of allelic specific primers were designed to distinguish the two HLA-C alleles of each individual with frequency over 0.96%in 835 cases of Han population.Among 55%of the liver cancer tissues ,the expression levels of the two HLA-C alleles from the same tumor tissue changes differently compared to that of the relevant non-tumor tissue.Conclusion:This study provides method for HLA-C allele expression level analysis of Han population and each HLA-C allele expression level is inconsistent of liver cancer tissue .
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Objective: To analyze the expression of terpene synthases of Aquilaria sinensis, an endangered south medicine, and to predict the influences of the environmental factors and stresses on the synthesis of terpene in A. sinensis. Methods: Two-year old seedlings and calli were treated by different stresses. The gene expression patterns of terpene synthases were analyzed by real-time qPCR. Results: Wounding and cauterizing could induce the transcriptional expression of terpene synthases in the stems, and the effect of cauterizing was more significant. Low temperature inhibited the transcription of terpene synthases. In calli, methyl jasmonate (MeJA) treatment had the most effective induction. Conclusion: Both wounding and cauterizing could induce the synthesis of terpenes in stems, while the cauterizing treatment might have better effect. Low temperature has negative influence on agarwood formation. For calli, different treatments could induce the terpene synthesis, while MeJA has the best efficacy.
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BACKGROUND: Accurate quantitative testing of HBV DNA is very important for choosing antiviral treatment targets and evaluating treatment response in chronic HBV patients. We evaluated the performance of LG AdvanSure HBV Real-Time QPCR kit (LG) utilizing real-time quantitative PCR. METHODS: The LG kit was conducted for 201 chronic hepatitis patients undergoing treatment at the Korea University Ansan hospital and 48 normal control volunteers. The precision, limit of detection, sensitivity, and specificity of LG Kit were evaluated. Correlation analysis was done with Abbott Real Time HBV kit (Abbott) and the Cobas Amplicor HBV Monitor kit (Cobas) and the concordances rate of the three methods were calculated. RESULTS: The LG assay showed linear range of detection from 10(2) to 10(6) and coefficient of variation (CV) was 1.10~0.52% at > or =1,000 IU/mL and 1.19% at 100 IU/mL. The coefficient of determination for precision analysis was 0.997. The limit of detection for detection of 95% of positive samples was 9.71 IU/mL (54.4 copies/mL). In 201 clinical samples, the log HBV DNA/ml showed good correlation between Roche vs Abott, Roche vs LG and Abott vs LG, respectively (n=105, 108, 133, r2=0.91, 0.89, 0.94, P0.05). CONCLUSION: LG AdvanSure HBV Real-Time QPCR kit showed outstanding precision, linearity, limit of detection, good correlation with previous methods, and is a valuable tool in treatment monitoring of chronic HBV infections.
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Humans , DNA , Hepatitis B , Hepatitis, Chronic , Korea , Limit of Detection , Organothiophosphorus Compounds , Sensitivity and SpecificityABSTRACT
ObjectiveTo investigate the expression of miR-155 in whole blood of patients with breast cancer and explore the possibility of miR-155 in whole blood as a marker of beast cancer. Methods65 cases (breast cancer:47 cases, non-breast cancer:18 cases) in Thyroid and Breast Surgery Department of No.1 People's Hospital of Huai'an from Dec 2010 to Apr 2011 were enrolled according to the selected criteria.Two milliliters anticoagulant blood were sampled to isolate total RNA.The expression level of miR-155 in whole blood was measured by real-time quantitative polymerase chain reaction (real-time qPCR)analysis.The relationship between the expression level of miR-155 in whole blood and the clinical pathological fearutres was analyzed.ResultsThe expression level of miR-155 in breast cancer patients was up-regulated compared with that in non-breast cancer patients(P ＜ 0.05).The expression level of miR-155 in patients with positive lymph nodes was up-regulated compared with that in patients with negative lymph nodes( P ＜ 0.05).The expression level of miR-155 in stage Ⅲ breast cancer was up-regulated compared with that in stage Ⅰ & Ⅱ breast cancer( P ＜ 0.05 ).The expression level of miR-155 in patients with positive ER and PR was down-regulated compared with that in patients with negative ER and PR breast cancer.Conclusion The study demonstrates that the expression of miR-155 in whole blood is related to clinical pathological features of patients with breast cancer and can be used as a potential marker of breast cancer.
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Objective To detect the expression of DNA damage response genes induced by radiation in human peripheral blood lymphocyte,and to explore the new biomarkers of radiation.Methods The human peripheral blood cells were irradiated to X-rays at different doses of 0,1,2,3,4,and 5 Gy.The quantitative real.time qPCR wag used to detect the expressions of cyclin-dependent kinase inhibitor l a gene(Cdknl a)and growth arrest and DNA damage inducible gene(Gadd45a)in lymphoeytes at 4 and 24 h post-irradiation,respectively.The method of CB mieronucleus was used to determine the change of micronucleus ratio.Results The expression of Cdknl a in peripheral blood lymphocytes wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy.reached the peak at 4 Gy and began to decrease at 5 Gy,which showed a dose-dependent manner(r=0.946,0.975,P<0.05).Similarly,the expression of Gadd45α in human peripheral blood lymphocytes was also increased significantly at 4 and 24 h post-irradiation to 0-5 Gy in a dose-dependent manner,while the expression of Gadd45a at 4 h wag higher than that at 24 h(r=0.936,0.797,P<0.05).The ratio of micronuclei wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy(r=0.990,0.984,P<0.05).Conciusions Cdknl a and Gadd45α expression could be increaged significandy at 4 and 24 h post-irradiation to 0-5 Gy,showing a good linear relationship.which might be candidate for radiation biological dosimeter.