Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC,

Despite numerous studies reporting deregulated microRNA (miRNA) and gene expression patterns in clear cell renal cell carcinoma (ccRCC), no direct comparisons have been made to its presumed normal counterpart: the renal proximal tubular epithelial cells (PTECs). The aim of this study was to determine the miRNA expression profiles of 10 ccRCC-derived cell lines and short-term cultures of PTEC and to correlate these with their gene expression and copy-number profiles. Using microarray-based methods, a significantly altered expression level in ccRCC cell lines was observed for 23 miRNAs and 1630...

MicroRNAs (miRNAs) play important roles in development, differentiation, homeostasis, and also in diseases such as lymphoma. This chapter describes methods to study the role of miRNAs in lymphoma. First, we describe a multiplex RT reaction followed by qPCR that can be used to determine differential expression of candidate miRNAs. Second, we provide a protocol for stable overexpression of miRNAs using lentiviral-based ectopic expression systems. Third, we describe a straightforward assay to determine whether the candidate miRNA may function as an oncogene or a tumor suppressor gene in lymphomagenesis.

MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We describe here a straightforward method to generate retroviral miRNA sponge constructs using a single directional ligation reaction. This approach allows generation of sponges containing more than 20 miRNA binding sites. We provide a basis for the design of the sponge constructs with...

Apoptosis induced by B-cell receptor (BCR) signaling is critical for antigen-driven selection, a process critical to tolerance and immunity. Here, we examined the roles of microRNAs (miRNAs) in BCR signaling-induced apoptosis using the widely applied WEHI-231 model. Comparison of miRNA levels in BCR-stimulated and -unstimulated cells revealed that 39 miRNAs were differentially expressed upon stimulation of the BCR. Importantly, stimulation in the presence of anti-CD40 antibodies, which rescues cells from BCR-induced apoptosis, prevented most changes in miRNA expression. Ectopic expression of mir-150 and mir-181a1b1, miRNAs that were upregulated...