The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).

In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo.

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. We present data in this study showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point - the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance.

Netrin-guided accessory cell morphogenesis dictates the dendrite orientation and migration of a Drosophila sensory neuron.

Accessory cells, which include glia and other cell types that develop in close association with neurons, have been shown to play key roles in regulating neuron development. However, the underlying molecular and cellular mechanisms remain poorly understood. A particularly intimate association between accessory cells and neurons is found in insect chordotonal organs. We have found that the cap cell, one of two accessory cells of v'ch1, a chordotonal organ in the Drosophila embryo, strongly influences the development of its associated neuron. As it projects a long dorsally directed cellular extension, the cap cell reorients the dendrite of the v'ch1 neuron and tows its cell body dorsally. Cap cell morphogenesis is regulated by Netrin-A, which is produced by epidermal cells at the destination of the cap cell process. In Netrin-A mutant embryos, the cap cell forms an aberrant, ventrally directed process. As the cap cell maintains a close physical connection with the tip of the dendrite, the latter is dragged into an abnormal position and orientation, and the neuron fails to undergo its normal dorsal migration. Misexpression of Netrin-A in oenocytes, secretory cells that lie ventral to the cap cell, leads to aberrant cap cell morphogenesis, suggesting that Netrin-A acts as an instructive cue to direct the growth of the cap cell process. The netrin receptor Frazzled is required for normal cap cell morphogenesis, and mutant rescue experiments indicate that it acts in a cell-autonomous fashion.

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo.

BACKGROUND: Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. RESULTS: We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. CONCLUSION: We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion.

Mu opioid receptors are expressed on radial glia but not migrating neuroblasts in the late embryonic mouse brain.

Mu opioid receptor ligands such as morphine and met-enkephalin are known to modulate normal brain development by perturbing gliogenesis and inhibiting neuronal proliferation. Surprisingly, the distribution of the mu opioid receptor (MOR) in the embryonic brain, especially in proliferative regions, is poorly defined and subject to conflicting reports. Using an immunohistochemical approach, we found that MOR protein was expressed in the neuroepithelia of the lateral ventricles, third ventricle, and aqueduct within the late embryonic (E15.5 and E18.5) mouse brain. In contrast to the ventricular neuroepithelia, the proliferative external granule layer of the embryonic cerebellum did not express MOR protein, although the Purkinje cell layer did. Within the ventricular neuroepithelium, GLAST-positive radial glia that incorporate BrdU expressed MOR, while migrating neuroblasts (doublecortin-positive) do not. BrdU labeling of proliferating cells showed an anterior to posterior gradient of proliferation (P<0.05), while an opposing posterior to anterior gradient of MOR expression (P<0.05) was found. The localization of MOR immunoreactivity within the embryonic ventricular neuroepithelia is consistent with a role for opioids in modulating neurogenesis.

Comparative quantitation of mRNA expression in the central nervous system using fluorescence in situ hybridization.

In situ hybridization to messenger RNA (mRNA) in complex tissues, such as the brain, allows the localization of gene expression to functionally distinct regions. It has been difficult to measure relative changes in gene expression within these regions because of the poor cellular resolution afforded by radioactively labeled probes and problems associated with densitometric analysis by counting silver grain deposition. Fluorescence in situ hybridization, using probes directly labeled with dyes that exhibit high quantum yield, provides both high-resolution localization of mRNA and high sensitivity for detection of hybridized probe. Digital image capture of fluorescence is readily amenable to densitometric analysis, thereby allowing relative quantification of mRNA expression in single cells or discrete brain nuclei. In this chapter, we describe protocols suitable for measuring relative changes in gene expression within individual cells of brain sections mounted on glass slides.

Abundant expression of mu and delta opioid receptor mRNA and protein in the cerebellum of the fetal, neonatal, and adult rat.

Opioid receptor proteins and mRNAs have been localized to a variety of regions within the rat brain. It is generally accepted that within the lobes of the rat cerebellum, only delta opioid receptor (DOR) is expressed. This is in contrast to that observed in humans and rabbits which express both mu opioid receptor (MOR) and DOR. In this study, we report detection of MOR as well as DOR protein by immunohistochemical localization, and mRNA by fluorescent in situ hybridization (FISH) within Purkinje cells (PK) and the granular layer of neonatal (P6) and adult rat cerebellum. Expression of MOR mRNA was also detected within cells of the molecular layer, but at lower levels than those seen within the PK cells. Abundant expression of MOR and DOR mRNA was detected in the external germinal layer of the immature cerebellum of the fetal (E16) rat, supporting a role for MOR and DOR in regulating neurogenesis of the cerebellum. In addition, using exon-specific cRNA probes, exons 1 and 4, which are both found in the MOR-1 splice variant mRNA, were detected in PK cells in the cerebellum and also within deep cerebellar nuclei in the adult.