Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction

MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.

The functions of animal microRNAs

Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.

/pdf/microrna-expression-profiles-classify-human-cancers-43jqfv3rwu.pdf

MicroRNA expression profiles classify human cancers

MicroRNA (miRNA ) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.

MicroRNA signatures in human cancers

Publisher Summary This chapter describes the use of a novel indicator gene, producing a secreted form of the human enzyme placental alkaline phosphate, that believes offers a number of advantages. These include very high stability, efficient secretion by all cells tested, and the availability of a simple, inexpensive, and highly quantitative assay that does not require any unusual equipment or reagents. A major focus of the current molecular biological research is the identification and functional dissection of the cis-acting sequences and trans-acting factors that regulate eukaryotic gene expression in vivo. A common method of addressing this question is to link the regulatory sequence of interest, e.g., an inducible promoter/enhancer element, to a reporter gene. Several alternate or complementary reporter gene systems have been proposed. Among the more prevalent are the genes for the intracellular enzymes β-galactosidase, luciferase and the secreted protein human growth hormone. This chapter describes SEAP, a novel indicator gene producing secreted alkaline phosphatase. The SEAP gene has several advantages when compared to CAT or to other prevalent reporter genes.

Secreted placental alkaline phosphatase as a eukaryotic reporter gene.

Although a great deal is known about the regulation of gene expression in terms of transcription, relatively little is known about the modulation of pre-mRNA processing. In this study, we exploited a genetically regulated system, human immunodeficiency virus type 1 (HIV-1) and its trans-activator Rev, to examine events that occur between the synthesis of pre-mRNA in the nucleus and the translation of mRNA in the cytoplasm. Unlike the majority of eukaryotic pre-mRNAs whose introns are efficiently recognized and spliced prior to nucleocytoplasmic transport, HIV-1 mRNAs containing functional introns must be exported to the cytoplasm for the expression of many viral proteins. Using human T cells containing stably integrated proviruses, we demonstrate that such incompletely spliced viral mRNAs are exported to the cytoplasm only in the presence of the Rev trans-activator. In the absence of Rev, these intron-containing RNAs are sequestered in the T-cell nucleus and either spliced or, more commonly, degraded. Because Rev does not inhibit the expression of fully spliced viral mRNA species in T cells, we propose that Rev, rather than inhibiting viral pre-mRNA splicing, is acting here both to prevent the nuclear degradation of HIV-1 pre-mRNAs and to induce their translocation to the cytoplasm. Taken together, these findings indicate that the cellular factors responsible for the nuclear retention of unspliced pre-mRNAs, although most probably splicing factors, do not invariably commit these RNAs to productive splicing and can, instead, program such transcripts for degradation.

Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes.

Analyses of small RNA expression profiles have revealed that several DNA viruses—including particularly, herpesviruses—express high levels of multiple viral microRNAs (miRNAs) in infected cells. Here, I review our current understanding of how viral miRNAs influence viral replication and pathogenesis and discuss how viruses reshape the pattern of cellular miRNA expression. Indeed, viruses are now known to both activate and repress the expression of specific cellular miRNAs, and disrupting this process can perturb the ability of viruses to replicate normally. In addition, it is now clear that virally encoded miRNAs play a key role in inhibiting antiviral innate immune responses and can also promote cell transformation in culture. While our understanding of how viruses interact with miRNAs remains somewhat rudimentary, it is nevertheless already clear that these interactions can play a critical role in mediating viral pathogenesis and therefore may represent novel and highly specific targets for therapeutic intervention.

/pdf/viruses-and-micrornas-riscy-interactions-with-serious-drcyl32wjh.pdf

Viruses and microRNAs: RISCy interactions with serious consequences

The HIV-1 Tat protein: an RNA sequence-specific processivity factor?

RNA interference (RNAi) provides a powerful technique for the derivation and analysis of loss-of-function phenotypes in vertebrate cells, where alternative approaches are either arduous or frequently ineffective RNAi, however, is not always totally specific, and confirming the specificity, and hence the validity, of data obtained using RNAi therefore forms an essential component of experiments that rely on this technique Here I briefly review the potential pitfalls associated with RNAi, and then suggest experimental approaches that can be used to maximize the specificity of RNAi or to confirm that the data obtained are indeed valid

Bryan R. Cullen

Papers

Secreted placental alkaline phosphatase as a eukaryotic reporter gene.

Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes.

Viruses and microRNAs: RISCy interactions with serious consequences

The HIV-1 Tat protein: an RNA sequence-specific processivity factor?

Enhancing and confirming the specificity of RNAi experiments.