Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

BACKGROUND: The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields. RESULTS: A time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach. CONCLUSIONS: It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times

Characterization of variable EST SSR markers for Norway spruce (Picea abies L.)

<p>Abstract</p> <p>Background</p> <p>Norway spruce is widely distributed across Europe and the predominant tree of the Alpine region. Fast growth and the fact that timber can be harvested cost-effectively in relatively young populations define its status as one of the economically most important tree species of Northern Europe. In this study, EST derived simple sequence repeat (SSR) markers were developed for the assessment of putative functional diversity in Austrian Norway spruce stands.</p> <p>Results</p> <p>SSR sequences were identified by analyzing 14,022 publicly available EST sequences. Tri-nucleotide repeat motifs were most abundant in the data set followed by penta- and hexa-nucleotide repeats. Specific primer pairs were designed for sixty loci. Among these, 27 displayed polymorphism in a testing population of 16 <it>P. abies </it>individuals sampled across Austria and in an additional screening population of 96 <it>P. abies </it>individuals from two geographically distinct Austrian populations. Allele numbers per locus ranged from two to 17 with observed heterozygosity ranging from 0.075 to 0.99.</p> <p>Conclusions</p> <p>We have characterized variable EST SSR markers for Norway spruce detected in expressed genes. Due to their moderate to high degree of variability in the two tested screening populations, these newly developed SSR markers are well suited for the analysis of stress related functional variation present in Norway spruce populations.</p

Subacute AMD3100 Treatment Is Not Efficient in Neonatal Hypoxic-Ischemic Rats

Background and Purpose: Despite the advances in treating neonatal hypoxic-ischemic encephalopathy (HIE) with induced hypothermia, the rates of severe disability are still high among survivors. Preclinical studies have indicated that cell therapies with hematopoietic stem/progenitor cells could improve neurological outcomes in HIE. In this study, we investigated whether the administration of AMD3100, a CXCR4 antagonist that mobilizes hematopoietic stem/progenitor cells into the circulation, has therapeutic effects in HIE. Methods: P10 Wistar rats of both sexes were subjected to right common carotid artery occlusion or sham procedure, and then were exposed to hypoxia for 120 minutes. Two subcutaneous injections of AMD3100 or vehicle were given on the third and fourth day after HIE. We first assessed the interindividual variability in brain atrophy after experimental HIE and vehicle treatment in a small cohort of rats. Based on this exploratory analysis, we designed and conducted an experiment to test the efficacy of AMD3100. Brain atrophy on day 21 after HIE was defined as the primary end point. Secondary efficacy end points were cognitive (T-water maze) and motor function (rotarod) on days 17 and 18 after HIE, respectively. Results: AMD3100 did not decrease the brain atrophy in animals of either sex. Cognitive impairments were not observed in the T-water maze, but male hypoxic-ischemic animals exhibited motor coordination deficits on the rotarod, which were not improved by AMD3100. A separate analysis combining data from animals of both sexes also revealed no evidence of the effectiveness of AMD3100 treatment. Conclusions: These results indicate that the subacute treatment with AMD3100 does not improve structural and functional outcomes in a rat HIE model. </jats:sec

Augmenting rehabilitation robotics with spinal cord neuromodulation: A proof of concept

Rehabilitation robotics aims to promote activity-dependent reorganization of the nervous system. However, people with paralysis cannot generate sufficient activity during robot-assisted rehabilitation and, consequently, do not benefit from these therapies. Here, we developed an implantable spinal cord neuroprosthesis operating in a closed loop to promote robust activity during walking and cycling assisted by robotic devices. This neuroprosthesis is device agnostic and designed for seamless implementation by nonexpert users. Preliminary evaluations in participants with paralysis showed that the neuroprosthesis enabled well-organized patterns of muscle activity during robot-assisted walking and cycling. A proof-of-concept study suggested that robot-assisted rehabilitation augmented by the neuroprosthesis promoted sustained neurological improvements. Moreover, the neuroprosthesis augmented recreational walking and cycling activities outdoors. Future clinical trials will have to confirm these findings in a broader population

Augmenting rehabilitation robotics with spinal cord neuromodulation : a proof of concept

Rehabilitation robotics aims to augment activity during training in order to reorganize the nervous system and thus improve neurological recovery. The consequence is that the amount of activity determines the extent of the recovery. In people with spinal cord injury (SCI), the absence of residual activity restricts the ability of robotically-assisted motion to augment activity. To address this limitation, we promoted robust activity of lower limb muscles with an implanted spinal cord neuroprosthesis. We developed wireless technologies and closed-loop control algorithms to resolve the challenge of ensuring rigorous synchronization between the neuroprosthesis and the actuation of various robotic devices commonly used to support rehabilitation across the continuum of care. These technologies are device-agnostic and designed for seamless implementation by non-expert users. Preliminary evaluations in 5 participants with SCI showed that the neuroprosthesis enabled well-organized patterns of muscle activity during robot-assisted walking and cycling. A proof of concept study also suggested that robot-assisted rehabilitation augmented by the neuroprosthesis promotes neurological improvements that persist with the neuroprosthesis turned off. We finally illustrate the possibility to augment recreational walking and cycling activities outdoors with the neuroprosthesis. Sufficiently powered clinical trials will next have to establish the safety and efficacy of the synergy between rehabilitation robotics and spinal cord neuromodulation to improve the neurological recovery of people with SCI

Augmenting rehabilitation robotics with spinal cord neuromodulation : a proof of concept

Rehabilitation robotics aims to promote activity-dependent reorganization of the nervous system. However, people with paralysis cannot generate sufficient activity during robot-assisted rehabilitation and, consequently, do not benefit from these therapies. Here, we developed an implantable spinal cord neuroprosthesis operating in a closed loop to promote robust activity during walking and cycling assisted by robotic devices. This neuroprosthesis is device agnostic and designed for seamless implementation by nonexpert users. Preliminary evaluations in participants with paralysis showed that the neuroprosthesis enabled well-organized patterns of muscle activity during robot-assisted walking and cycling. A proof-of-concept study suggested that robot-assisted rehabilitation augmented by the neuroprosthesis promoted sustained neurological improvements. Moreover, the neuroprosthesis augmented recreational walking and cycling activities outdoors. Future clinical trials will have to confirm these findings in a broader population

Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5\u2032 ends that vary in size. Methods to identify the 5\u2032 ends of transcripts will facilitate the discovery of new promoters and 5\u2032 ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano\u2013cap analysis of gene expression (nanoCAGE), a method that captures the 5\u2032 ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5\u2032 ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome