ABSTRACT
Objective: In spite of accumulating information about pathological aspects of sulfur mustard [SM], the precise mechanism responsible for its effects is not well understood. Circulating microRNAs [miRNAs] are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims
Materials and Methods: In this case and control experimental study, using quantitative real-time polymerase chain reaction [qRT-PCR], we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war [1980-1988] and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle [Cq] method were employed to find the least variable reference gene
Results: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that
Conclusion: We demonstrate that non-miRNA reference genes have the least stability in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers
ABSTRACT
Human alpha 1-antitrypsin [AAT] cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZ?-AAT, human AAT was expressed in the yeast Pichia pastoris [P.pastoris] in a secretary manner and under the control of inducible alcohol oxidase 1 [AOX1] promoter. The amount of AAT protein in medium was measured as 60 mg/l 72 hr after induction with methanol. Results indicated the presence of protease inhibitory function of the protein against elastase. Purification was done using His-tag affinity chromatography. Due to the different patterns of glycosylation in yeast and human, the recombinant AAT showed different SDS-PAGE patterns compared to that of serum-derived AAT while pI shifted from 4.9 in native AAT compared to 5.2 in recombinant AAT constructed in this study
ABSTRACT
Salmonella enterica serovar Enteritidis which involves poultry is transmitted by food. The aim of this survey was to optimize PCR method in order to detect Salmonella enteritidis in poultry products. In this basic research, 80 samples [40 broiler meat and 40 egg samples] were obtained from distribution centers in Karaj and suburb. Following the DNA extraction of the samples, PCR was optimized and performed using standard strain of Salmonella enteritidis [RTCC1621] as positive control and primers against the flagella coding sequence of Salmonella Enteritidis genome. The analysis of the PCR products by agarose gel electrophoresis indicated the amplification of 250 bp segments in 16 out of 40 [40%] broiler meat and 9 out of 40 [23%] egg samples. The sensitivity of the PCR at the DNA level was found to be 1pg and the specificity of the PCR was determined using 6 other entric Gram negative bacteria and found to be positive only for Salmonella enteritidis. This study confirmed that PCR provides sensitive, specific and rapid approach for detection of Salmonella enteritidis in food samples
Subject(s)
Animals , Polymerase Chain Reaction , Poultry Products/microbiologyABSTRACT
Embryonic stem cells [ESCs] are pluripotent, self-renewing cells. These cells can be used in applications such as cell therapy, drug discovery, disease modelling, and the study of cellular differentiation. In this experimental study embryonic stem cells cultured in the laboratory and were amplified. Total RNA was extracted from cells and converted to cDNA. The replication factor Oct3/4 gene was amplified by reverse transcription-polymerase chain reaction [RT-PCR] and cloned into the pTZ57R/T vector. Legated product had been transformed into susceptible bacteria and transformed bacteria were screened on a selective medium. Plasmids extracted from bacteria and enzyme digestion to confirm the sequencing was performed. Results of enzyme digestion were sequenced. Cloned gene can prepare a gene cassette to produce stem cells from somatic cell
ABSTRACT
Pneumoviruses are responsible for significant respiratory disease in their hosts and represent a major problem for human and animal health. Pneumoviruses are members of the family Paramyxoviridae, subfamily Pneumovirinae and the virus particles consist of a negative-sense, nonsegmented RNA genome within a helical nucleocapsid structure enveloped in a lipid membrane derived from the host cell. Over the past four decades much work has extended our understanding of the molecular biology and pathogenesis of pneumoviruses but despite this only limited treatments and prophylaxis are available. The human pathogen, respiratory syncytial virus [hRSV] which belongs to the genus of Pneumovirus is the best characterized of the subfamily. HRSV is the major cause of hospitalisation of very young children with respiratory disease worldwide. No vaccine is available though new treatments offer some respite for children in the highest risk groups, the immunocompromised and children with congenital heart disease. The recently discovered human pathogen human metapneumovirus [hMPV] belongs to the genus Metapneumovirus and recent data indicates that this virus is second only to hRSV in terms of disease impact. The pneumoviruses also include agents of veterinary importance such as bovine respiratory syncytial virus [bRSV], ovine and caprine RSV, and pneumonia virus of mice [PVM: all in the genus Pneumovirus] and avian metapneumovirus [APV: genus Metapneumovirus]. The development of reverse genetics systems for negative strand RNA viruses has opened the possibility of manipulating the virus genomes to identify genes involved in pathogenesis and to explore the biological consequences of specific mutations. This information is informing the rational design of new vaccines. These plasmid-based systems have shown that for all paramyxoviruses the N, P and L proteins are necessary and sufficient for RNA replication. However, the pneumoviruses differ from the other family members in that fully efficient transcription from the virus genome requires the presence of an additional protein encoded by the M2 gene. The present article reviews pneumovirus biology and molecular genetics including a discussion of current concepts of Pneumovirus reverse genetics