Spatially revealed roles for lncRNAs in Drosophila spermatogenesis, Y chromosome function and evolution.

Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, we initiated a genome-wide analysis of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

Reconsenting paediatric research participants for use of identifying data.

When a minor research participant reaches the age of majority or the level of maturity necessary to be granted legal decision-making capacity, reconsent can be required for ongoing participation in research or use of health information and banked biological materials. Despite potential logistical concerns with implementation and ethical questions about the trade-offs between maximising respect for participant agency and facilitating research that may generate benefits, reconsent is the approach most consistent with both law and research ethics.Canadian common law consent requirements are expansive and likely compel reconsent on obtaining capacity. Common law doctrine recognises that children are entitled to decision-making authority that reflects their evolving intelligence and understanding. Health consent legislation varies by province but generally either compels reconsent on obtaining capacity or delegates the ability to determine reconsent to research ethics boards. These boards largely rely on the Canada's national ethics policy, the Tri-Council Policy Statement, which states that, with few exceptions, reconsent for continued participation is required when minors gain capacity that would allow them to consent to the research in which they participate. A strict interpretation of this policy could require researchers to perform frequent capacity assessments, potentially presenting feasibility concerns. In addition, Canadian policy and law are generally consistent with the core principles of key international ethical standards from the United Nations and elsewhere.In sum, reconsent of paediatric participants upon obtaining capacity should be explicit and informed in Canada, and should not be presumed from continued participation alone.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

The New RNA World: Growing Evidence for Long Noncoding RNA Functionality.

The past decade has seen a major increase in the study of noncoding RNAs (ncRNAs). However, there remains a great deal of confusion and debate over the levels of functionality and mechanisms of action of the majority of these new transcripts. This Opinion article addresses several of these issues, focusing particularly on long ncRNAs (lncRNAs). We reemphasize the unique abilities of RNAs to form myriad structures as well as to interact with other RNAs, DNA, and proteins, which provide them with unique and powerful abilities. One of these, the ability to interact sequence specifically with DNA, has been largely overlooked. Accumulating evidence suggests that evolution has taken advantage of RNA's properties via the rapid acquisition of new noncoding genes in testes, with subsequent gains of function in other tissues. This amplification process appears to be one of the major forces driving metazoan evolution and diversity.