What is it used for?Small RNA extraction is the first step prior to proceeding for microRNA RT-PCR. Before performing small RNA isolation from total RNA (extracted in previous section), two more steps are required: 1. DNase treatment and 2. Agarose gel electrophoresis, which are respectively crucial for eliminating of DNA contamination and to check the extracted RNA quality.

Background: Nanosilver (NS) contains silver nanoparticles to control infections delivered from bacteria. The NS compounds as well as being toxic to prokaryotes are highly toxic to mammalian cells. The present study was evaluated the effect of colloidal NS on leydig cells glucose transporter type I (GLUT I) expression, serum level of testosterone, germinal cells RNA damage and spermiogenesis process.Materials and Methods: Twenty four mature male mice were divided into four groups (n=6) as test and control sham groups. The animals in test groups received the colloid NS in doses of 0.5, 1 and 5 mg/kg and the animals in control-sham group received saline intraperitoneally, during 34 consecutive days. The immunohistochemical and Epi-fluorescent analyses were conducted to evaluate the GLUT I expression and germinal cells RNA damage, respectively. The serum testosterone level was assessed by RIA method. The spermiogenesis index was investigated.Results: The GLUT I expression significantly (p<0.05) decreased in NS-administrated animals. Accordingly, the high dose received mice were manifested with lowest GLUT I on leydig cells. The serum level of testosterone decreased depending on dose. The germinal cells were exhibited with severe RNA damage accomplished with remarkable reduction in the percentage of seminiferous tubules with positive spermiogenesis index.Conclusion: Our data suggest that NS partly by reducing GLUT I expression on the leydig cells membrane down-regulates the cells glucose intake. Therefore, the leydig cells loss their physiologic ability to synthesis testosterone. Ultimately, the induced impairment leads to severe RNA damage in germinal cells which negatively impacts the spermiogenesis process.

Background: Various fixation and permeabilization techniques have been de-veloped for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells.Methods: HeLa cells were fixed in 2% paraformaldehyde. A variety of deter-gents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeab-ilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets.Results: In comparison with other methods, maximum cell frequency in per-centage and fluorescent intensity (M1=2.1%, M2=97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p=0.001).Conclusion: Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized.

Background: RNA interference (RNAi) is a phenomenon uses double-stranded RNA (dsRNA) to specifically inhibit gene expression. The non-specific silencing caused by interferon response to dsRNA in mammalian cells limits the potential of utilizing RNAi to study gene function. Duplexes of 21-nucleotide short interfering dsRNA (siRNA) inhibit gene expression by RNAi. In some organisms, siRNA can also function as a primer converting mRNA into dsRNA that are further cleaved to produce more siRNA. This activity involves the enzyme RNA-dependent RNA polymerase (RdRP). There are no known RdRP involved in RNAi in mammals. By using an RdRP from Caenorhabditis elegance named ego-1, investigators intend to enhance RNAi effect in mammalian cells. The aims of this project were: 1) to investigate the efficiency of siRNA to enhanced green fluorescent protein (eGFP) gene silencing and 2) to enhance the RNAi effect. Methods: We used a vector-based siRNA to target eGFP. Also we used a vector expressing ego-1 to test for a possible amplification effect of RNAi. The expression of eGFP in the cells was detected by using fluorescent microscopy, flowcytometry and Western-blotting. Results: Transfection of the plasmid into P19 cells significantly decreased eGFP fluorescence. In addition, eGFP protein was reduced. Preliminary data suggested that the presence of ego-1 enhanced the RNAi effect. Conclusion: The results indicated that use of hairpin siRNA expression vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells. Also, introducing RdRP enzyme to mammalian cells might amplify the RNAi effect in the cells. Iran.