Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.ResultsAn average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).ConclusionThe RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis

From farm to fork: lipid oxidation in fish products. A review

Lipid oxidation is a very complex and important event threatening the quality of foods especially of those containing highly unsaturated fats. Fish are the main source of polyunsaturated fatty acids that, unfortunately, are highly susceptible to degradation process, such as oxidation. Fish supply chain generally involves many steps and each of them together with their interaction might play a central role in muscle quality maintenance. From this review emerged that antioxidants supplementation diet can play a central role to limit the detrimental effects of stress (pre-slaughter or at killing) and storage. In this sense, lycopene shows the best antioxidant activity during stressful conditions while α-tocopherol acts preferentially in long-term frozen storage. Stress just before or at slaughter can greatly threaten flesh quality both immediately and after storage by inducing numerous metabolic pathways, that often involve the production of very reactive molecular species, such as hydroperoxides. A common operation such as bleeding can significantly reduce both reactive molecules and haemoglobin (Hb), which is recognised as a great pro-oxidant. Temperature and duration are two critical points of storage phase which has to be considered even by consumers. Frozen storage at very low temperatures (−30 °C, −40 °C) confirms to be the best storage practise. Finally, cooking can compromise aromatic profile of cooking fillets. Thus, feeding antioxidant, reducing stress both during pre-slaughter practise and at killing, good storage practises, if associated with an appropriate cooking method (low temperature, short time) seems to be the clues for preserving the fragile lipid fraction from farm to fork