A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA

<p>Abstract</p> <p>Background</p> <p>The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts.</p> <p>Results</p> <p>We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci.</p> <p>Conclusion</p> <p>We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.</p

The P4 promoter of the parvovirus minute virus of mice is developmentally regulated in transgenic P4-LacZ mice

AbstractActivation of the minute virus of mice (MVM) P4 promoter is a key step in the life cycle of the virus and is completely dependent on host transcription factors. Since transcription-factor composition varies widely in different cell types, there is the possibility that only some cell types in the host organism have the capacity to initiate expression from the P4 promoter and therefore that the promoter may be a factor in determining the tropism of MVM. In this study, the ability of various cell types to activate P4, independent of the other virus–host interactions, was examined in transgenic mouse lines bearing a β-galactosidase reporter sequence driven by the P4 promoter. It was found that lacZ was expressed during embryogenesis and in the adult in a cell-type-specific and differentiation-dependent pattern. The data are consistent with cell-type and stage-specific activation of the P4 promoter having a role in determining the host cell-type range of MVM. The ability of some parvoviruses to replicate in, and kill oncogenically transformed cells, and to destroy induced tumors in laboratory animals is the basis of recent approaches to use MVM-based vectors in cancer gene therapy. Since these vectors rely on the activation of the P4 promoter by the target tissues, understanding the promoter dependence on cell-type and differentiation status is important for their design and potential use

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA-0

<p><b>Copyright information:</b></p><p>Taken from "A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA"</p><p>http://www.biomedcentral.com/1471-2164/8/363</p><p>BMC Genomics 2007;8():363-363.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2134933.</p><p></p>bases (1), an AscI site (2), a 5 nt random sequence (3), and a 3' T residue (4). By design, priming should begin only at an A in the RNA template. . The non-phosphorylated lone-linker LL1 consists of the the two complimentary oligonucleotides LL1F and LL1R. It has one blunt end and one non-adhesive staggered end. LL1 can therefore ligate only to one strand of the cDNAs and in only one orientation. The remaining nick in the second strand is removed by preincubating the cDNAs before the first PCR reaction at 72°C for one minute to strip off the non-ligated strand of the linker and regenerate the sequence by extension from the 3' end of the cDNA (lower grey)

A Twist-Snail Axis Critical for TrkB-Induced Epithelial-Mesenchymal Transition-Like Transformation, Anoikis Resistance, and Metastasis▿ §

In a genomewide anoikis suppression screen for metastasis genes, we previously identified the neurotrophic receptor tyrosine kinase TrkB. In mouse xenografts, activated TrkB caused highly invasive and metastatic tumors. Here, we describe that TrkB also induces a strong morphological transformation, resembling epithelial-mesenchymal transition (EMT). This required TrkB kinase activity, a functional mitogen-activated protein kinase pathway, suppression of E-cadherin, and induction of Twist, a transcription factor contributing to EMT and metastasis. RNA interference (RNAi)-mediated Twist depletion blocked TrkB-induced EMT-like transformation, anoikis suppression, and growth of tumor xenografts. By searching for essential effectors of TrkB-Twist signaling, we found that Twist induces Snail, another EMT regulator associated with poor cancer prognosis. Snail depletion impaired EMT-like transformation and anoikis suppression induced by TrkB, but in contrast to Twist depletion, it failed to inhibit tumor growth. Instead, Snail RNAi specifically impaired the formation of lung metastases. Epistasis experiments suggested that Twist acts upstream from Snail. Our results demonstrate that TrkB signaling activates a Twist-Snail axis that is critically involved in EMT-like transformation, tumorigenesis, and metastasis. Moreover, our data shed more light on the epistatic relationship between Twist and Snail, two key transcriptional regulators of EMT and metastasis