The huge amount of transcript data produced by high-throughput sequencing requires the development and implementation of suitable bioinformatic workflows for their analysis and interpretation. These analysis workflows, including different modules, should be specifically designed also based on the sequencing platform (Roche 454, Illumina, SOLiD) and the nature of the data (polyA or total RNA fraction, strand specificity). In the case of cDNA obtained from a total RNA preparation, in addition to polyadenylated protein coding mRNAs, a great variety of transcript sequences can be obtained, including ribosomal RNAs, mitochondrial transcripts and a large variety of functional non coding RNAs (ncRNAs). To deal with these data the analysis workflow should include specific modules to distinguish ncRNAs fractions from the large number of other functional proteincoding transcripts. To this aim we developed an analysis pipeline that, given as input a large collection of reads (particularly from Roche 454), provides the expression profile at qualitative and quantitative level of human mtDNA, ribosomal RNAs, ncRNAs and protein coding mRNAs.

Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform.ResultsWe set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol.ConclusionWe tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that ?-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to ?-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1? (HIF1?) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1? inhibition by HIF1?, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1? stabilization, in fact, blunts the mitochondrial response to ?-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1? increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.