RT-qPCR gene expression analysis in zebrafish: Preanalytical precautions and use of expressed repetitive elements for normalization.

Gene expression analysis is increasingly important in many fields of biological research. Understanding patterns of expressed genes is assumed to provide insight into complex regulatory networks and can lead to the identification of genes relevant to specific biological processes, including disease. Among different techniques, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is currently regarded as the gold standard for targeted quantification of RNA gene expression, especially because of its high sensitivity, specificity, accuracy, and precision, and also because of its practical simplicity and processing speed. However, different critical factors can influence the outcome of RT-qPCR studies, including isolation of RNA, reverse transcription to cDNA, and data analysis. These factors need to be addressed in order to obtain biologically meaningful results. In this chapter, we describe how RT-qPCR can be used in a reliable way to successfully study differential gene expression in zebrafish. Hereby, we especially focus on how expressed repetitive elements can be employed as reference targets in zebrafish RT-qPCR studies and how they can further improve the quality of the data.

MYCN and ALKF1174L are sufficient to drive neuroblastoma development from neural crest progenitor cells.

Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALK(F1174L). For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycER(T)). Expression of MYCN or ALK(F1174L), one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycER(T) activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.

Percutaneous compression plating (PCCP) versus the dynamic hip screw for pertrochanteric hip fractures: preliminary results.

The percutaneous compression plate (PCCP) is a new implant for the minimally invasive treatment of pertrochanteric hip fractures that might reduce blood loss, wound problems and prevent devascularization of bone fragments. A quicker operation with minimal blood loss is better in the older patients. We performed a prospective, randomized clinical trial to compare the PCCP with the well-known dynamic hip screw (DHS). A total of 71 patients with an Evans type 1A-D pertrochanteric hip fractures were included. We measured the operation duration, blood loss, wound healing, complications, fracture healing and functional outcome. In total, 33 PCCP and 38 DHS were implanted. The mean operation times were 69.2 and 46.6 min for DHS and PCCP, respectively (P = 0.000). Blood transfusions were given in 24 DHS patients compared with six PCCP patients (P = 0.000). There were 27 haematomas in the DHS group and eight in the PCCP group (P = 0.000). There were no differences in fracture healing and the functional outcome between the two implants (P = 0.767, ns). Although this is a preliminary study with a relatively small number of patients and short follow-up, the PCCP seems similar to the DHS in relation to bone healing and stability, but with significant advantages for blood loss, soft tissue healing and operation time.