RESUMO
Understanding gene expression variations by using RNA transcript analysis methods during hepatic viral protein interactions with the IFN pathway in hepatic cell lines has recently gained importance. One of the most powerful techniques in gene expression quantification is quantitative real-time RT-PCR. Reference genes used as normalizer in this method may be affected across various experimental conditions or treatments. Hence, in the present study, the influence of IFN-treatment on the mRNA levels of common reference genes including ACTB, GAPDH, TBP, HPRT1 and HMBS was evaluated in Huh-7 or HepG[2] cell lines. Cells were treated with different concentrations of IFN- Then, using geNorm and NormFinder programs, we evaluated the expression stabilities of the above prominent reference genes in three sample groups that included each hepatic cell line and the total data sets. HPRT-1 and GAPDH were the most stable reference genes in the Huh-7 cell line, whereas ATCB, HMBS and GAPDH were the most stable in the HepG2 cell line. TBP was one of the least stable reference genes in the three studied groups. This investigation will provide appropriate reference genes for standardization of quantitative real-time PCR data in an IFN-?stimulated model of hepatocyte cell lines
RESUMO
Background: human rotavirus genotypes G1-G4, G9, P [4] and P [8] are major worldwide causes of acute gastroenteritis in children. Rotavirus genotype G1P [8] is predominant in many countries. In this study, the genotypic diversity of group A rotaviruses were detected in children <5 years of age who were treated for dehydration and diarrhea in Tehran, Iran from October 2004 to September 2008
Methods: a total of 700 stool specimens were collected from children and assessed for the presence of rotaviruses by the dsRNA-PAGE technique. G and P typing of the positive samples were performed by semi-nested multiplex RT-PCR
Results: rotaviruses were isolated in 19% of samples. A total of 14 rotavirus dsRNA different electrophoretypes were detected. The predominant genotype was G1 [76.3%], followed by G4 [11.5%], G8 [0.8%], P [4] [9.2%] and P [8] [66.4%], respectively. In mixed type samples, the majority were of genotype G1P [8] [53.4%], followed by G1P [4] [9.2%] and G4P [8] [4.6%]. Mixed types consisted of 3.1% of the total sample followed by G1G2/-P [1.5%], G1G4P [4] [0.8%] and G1G4P [8] [0.8%]
Conclusion: in this study, a high prevalence of the G1P [8] genotype was determined to be the cause of childhood gastroenteritis in Tehran, Iran. The sequence of G and P genotypes showed high levels of similarity to strains from other Asian countries. Our data will be useful for future vaccine formulation in Iran
RESUMO
Prostate cancer is one of the most common cancer in the developed countries. Most of cancer deaths are due to development of metastasis. Hence, prevention of metastasis is critical. Silibinin is a flavonoid component that inhibits cell proliferation and causes cell death of human prostate cancer. In this study, the expression of CD82 gene in PC-3 cells treated with escalating concentrations of silibinin was evaluated which can result in new view for prostate cancer therapy. In this study, PC-3 cells were treated with different concentrations of silibinin for 24h. The LD50 was determined. RNA was extracted by trizol, then cDNA was synthesized. Precise primers were designed for CD82 and GAPDH genes by specific software. Quantity of CD82 gene expression compare to GAPDH gene in different concentrations of silibilin was analyzed using very sensitive quantitative Real-time PCR. CD82 gene expression in PC-3 cells treated with 100, 150 and 200?g/ml of silibinin at 24h was increased by 1.97 +/- 0.26 [P<0.05], 3.00 +/- 0.26 and 3.43 +/- 0.43 [P<0.01], respectively. The results of quantitative Real-time PCR indicated that silibinin can probably decrease metastasis, by up-regulation of CD82 metastasis suppressor gene in PC-3 cells
RESUMO
In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26_seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIB-1 Monitor test. The results demonstrated that this technique could detect up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers [5x10[2]- 5x10[9]]. Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results [R[2] =0.95]. On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test [5x10[9] versus 7.5x10[5]]. This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis, Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than 3% and 4.5% accordingly. The above results, indicates that the new developed test can be a used in substitution of the commercial assay for quantitative analysis of HIV-1