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The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.Electronic supplementary materialThe online version of this article (10.1007/s00414-018-1817-5) contains supplementary material, which is available to authorized users.

MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene. Neuroblastoma (NB),1 a neoplasm that arises from embryonic neural crest tissue, is the second-most common solid pediatric tumor (1). MYCN is amplified in ϳ25% of primary NBs (2-4), and MYCN overexpression is frequently detected in NB tumors (5). Although the clinical significance of MYCN expression in NB tumors that lack MYCN amplification remains controversial (6 -10), a strong correlation between high levels of MYCN expression consequent to genomic amplification of this oncogene and aggressive disease is well established (3-5, 11). Laboratory experiments have further supported an important role for MYCN in determining NB phenotype. Ectopic overexpression of MYCN results in enhanced malignant growth (12, 13), whereas MYCN antisense studies performed by our laboratory and others have shown that down-regulation of MYCN in human NB is associated with a decrease in cellular proliferation and inhibited tumor cell growth in vitro (14 -16). Furthermore, a direct link between MYCN expression and the development of NB has been demonstrated in transgenic mice studies (17).Previously we examined MYCN regulation in N-(neuroblastic, tumorigenic) and S-type (substrate-adherent, non-tumorigenic) subclones (W-N and W-S) of the MYCN-amplified NB cell line NBL-W (18). In addition to distinct morphology and growth characteristics, the subclones have differential levels of MYCN expression despite having the same genomic MYCN copy number. We found that the disparity in steady-state levels of MYCN mRNA in the W-N and W-S cells was largely determined by differences in MYCN mRNA stability (19). Lazarova and co-workers (20) examined MYCN expression in other ...

Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/ instability by protein kinase A and focused our attention on the 3-untranslated region (3-UTR) of LDH-A mRNA. We have constructed various chimeric globin/ lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the halflife of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH- Analysis of the LDH 1 isoenzyme patterns in various cell types under a variety of physiologic conditions suggests complex regulatory mechanisms that determine specific isoenzyme expression (1-7). The LDH-A subunit, for instance, is subject to regulation by a number of different effector agents such as estrogen (3,8), epidermal growth factor (5), catecholamines (4, 9), phorbol ester (7), and c-Myc (10), which change the isoenzyme pattern almost exclusively in favor of the LDH-5 (A4) isoenzyme. The functional importance of these LDH isoenzyme shifts is generally attributed to a need for increased A subunitcontaining isoforms (such as LDH-4 or -5), which can derive more energy from the anaerobic pathway by reducing pyruvate to lactate. Investigations into the mechanism of LDH-A gene expression has identified two basic controls consisting of a transcription-regulatory cascade (4, 6, 11) and a mechanism that regulates the half-life of LDH-A mRNA (4, 12), both of which are major determinants of intracellular LDH-A mRNA levels.Messenger RNA turnover rates fluctuate over a wide range, and it is important to identify and characterize putative stability-regulating mRNA domains and their interacting factors that may be responsible for these functional effects. A great number of reports have demonstrated the existence of such domains and their trans-acting regulatory factors that are critical in determining the half-life of mRNA (13). Several of these studies indicate that the stability of some, but not all, mRNA is determined by specific cis-acting AU-rich domains located in the 3Ј-UTR. For example, a number of mRNAs such as cytokine, lymphokine and protooncogene mRNAs share a common sequence motif with a high content of A and U nucleotides in the 3Ј-UTR (14) and exhibit half-lives in the range of only a fraction of 1 h (15-18). In addition, attention has focused on modulation of mRNA stability in response to a variety of physiological signals. For instance, histone mRNA stability is regulated by the cell cycle (19) and intracellular iron levels control the stability of transferrin receptor mRNA (20,21). Moreover, manipulation of cells with several different effector agents can alter the steady-state level of mRNA during cell g...

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