A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

Background  

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence.     

Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study

Background  

RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.     

Characterization of bovine miRNAs by sequencing and bioinformatics analysis

Background  

MicroRNAs (miRNAs) are a family of ~22 nucleotide small RNA molecules which regulate gene expression by fully or partially binding to their complementary sequences in mRNAs or promoters. A large number of miRNAs and their expression patterns have been reported in human, mouse and rat. However, miRNAs and their expression patterns in live stock species such as beef cattle are not well studied.     

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background  

The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.     

Altered microRNA expression in patients with non-obstructive azoospermia

Background  

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are indicated to play essential roles in spermatogenesis. However, little is known about the expression patterns or function of miRNAs in human testes involved in infertility.     

Detecting microRNA activity from gene expression data

Background  

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions.     

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression

Background  

RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.     

miRAS: a data processing system for miRNA expression profiling study

Background  

The study of microRNAs (miRNAs) is attracting great considerations. Recent studies revealed that miRNAs play as important regulators of gene expression and some even as cancer players or inhibitors. Many studies try to discover new miRNAs and reveal the miRNA expression profile in cancer using a SAGE-based total RNA clone method. However, the data processing of this method is labor-intensive with several different biological databases and more than ten data processing steps involved.     

Measuring microRNAs: Comparisons of microarray and quantitative PCR measurements,and of different total RNA prep methods

Background  

Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used.     

miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

Background  

MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.