Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves

Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs

Neuregulin1/ErbB4-induced migration in ST14A striatal progenitors: calcium-dependent mechanisms and modulation by NMDA receptor activation

<p>Abstract</p> <p>Background</p> <p>A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence.</p> <p>Results</p> <p>RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca²⁺channels characterized by low Mg²⁺-sensitivity and low Ca²⁺-permeability, generating small, long-lasting currents. Ca²⁺-imaging experiments showed slow [Ca²⁺]_iincreases in 45% of the cells following 8 μM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca²⁺in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca²⁺]_i, in 99% of the cells. These intracellular Ca²⁺signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals.</p> <p>Conclusions</p> <p>In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca²⁺]_iincrease in ST14A neural progenitors; NRG1-induced migration is Ca²⁺-dependent and can be positively modulated by activation of the NMDA receptor.</p