Discovery of barley miRNAs through deep sequencing of short reads

Background: MicroRNAs are important components of the regulatory network of biological systems and thousands have been discovered in both animals and plants. Systematic investigations performed in species with sequenced genomes such as Arabidopsis, rice, poplar and Brachypodium have provided insights into the evolutionary relationships of this class of small RNAs among plants. However, miRNAs from barley, one of the most important cereal crops, remain unknown. Results: We performed a large scale study of barley miRNAs through deep sequencing of small RNAs extracted from leaves of two barley cultivars. By using the presence of miRNA precursor sequences in related genomes as one of a number of supporting criteria, we identified up to 100 miRNAs in barley. Of these only 56 have orthologs in wheat, rice or Brachypodium that are known to be expressed, while up to 44 appear to be specifically expressed in barley. Conclusions: Our study, the first large scale investigation of small RNAs in barley, has identified up to 100 miRNAs. We demonstrate that reliable identification of miRNAs via deep sequencing in a species whose genome has not been sequenced requires a more careful analysis of sequencing errors than is commonly performed. We devised a read filtering procedure for dealing with errors. In addition, we found that the use of a large dataset of almost 35 million reads permits the use of read abundance distributions along putative precursor sequences as a practical tool for isolating miRNAs in a large background of reads originating from other non-coding and coding RNAs. This study therefore provides a generic approach for discovering novel miRNAs where no genome sequence is available.Andreas W Schreiber, Bu-Jun Shi, Chun-Yuan Huang, Peter Langridge, Ute Bauman

Temperature Switch PCR (TSP): a gel-based molecular marker technique for investigating single nucleotide polymorphisms

Temperature Switch PCR (TSP) is a robust single-marker single nucleotide polymorphism (SNP) genotyping technique with broad applications in genetic studies of various organisms. The technique consists of a biphasic PCR with two sets of primers, a locus-specific set and a nested locus-specific set. The PCR products can be easily assessed for polymorphism based on different band sizes using agarose gel electrophoresis.Le Phuoc Thanh and Kelvin Kho

Clinimetric evaluation of a new overall disability scale in immune mediated polyneuropathies

Objectives: To determine the validity, reliability, and responsiveness of a new overall disability sum score in immune mediated polyneuropathies. Methods: Three impairment measures (MRC sum score, sensory sum score, grip strength (Vigorimeter)) and three disability scales (an overall disability sum score (ODSS), Hughes' functional scale (f score), Rankin scale) were assessed in a cross sectional group of 113 clinically stable patients (83 with Guillain–Barré syndrome, 22 with chronic inflammatory demyelinating polyneuropathy (CIDP), eight with a gammopathy related polyneuropathy). The ODSS was also used serially in 20 patients with recently diagnosed Guillain–Barré syndrome (n = 7) or CIDP (n = 13) and changing clinical conditions. Multiple regression studies were performed to compare the impact of impairment disturbances (independent variables) on the various disability scales (dependent variable). Results: Moderate to good construct validity (stable group: Spearman's rank test (absolute values), r = 0.41–0.79; longitudinal group: multiple correlation coefficient, R = 0.69–0.89; p < 0.006 for all associations) and reliability (intraclass correlation coefficient, R = 0.90–0.95; p < 0.0001) were demonstrated for the ODSS. Its SRM values were high (> 0.8), indicating good responsiveness. Impairment measures accounted for a higher variance proportion of the ODSS compared with the f score and Rankin (R = 0.64 v 0.56 and 0.45, respectively). Conclusions: All clinimetric requirements were met by the overall (arm and leg) disability sum score in immune mediated polyneuropathies. Its use is therefore suggested in evaluating immune mediated polyneuropathies