neuroscience peptides The second most stable reference

The second most stable reference gene in the present study comparing OA- and healthy BMSCs is IPO8. The IPO8 gene product is a member of the importin-β family responsible for the nuclear import of proteins (Dean et al., 2001). Furthermore, it is involved in cytoplasmic miRNA-guided gene silencing and nuclear Ago protein localization (Weinmann et al., 2009). IPO8 was identified in human differentiated primary preadipocytes and in human adipose tissue by using geNorm and NormFinder as a suitable reference gene for qRT-PCR studies (Hurtado del Pozo et al., 2010). Riemer et al. treated human keratinocyte cell lines with and without interferon-γ in qRT-PCR reference gene validation. Using geNorm and NormFinder they provide clear evidence that IPO8 is one of the most stably expressed genes in the explained experimental setup (Riemer et al., 2012). IPO8 was found to be the most suitable reference gene for lung (Nguewa et al., 2008), colon (Sørby et al., 2010), and neurological (Kreth et al., 2010) tumors. CASC3 being the third most stably expressed gene in our study, has not been recommended as reference gene before. CASC3, also known as metastatic lymph node 51 (MLN51), encodes a nucleocytoplasmic core protein of the exon junction complex (EJC) and is further associating with spliced mRNA in the cell nucleus and cytoplasm of mammalian neuroscience peptides (Degot et al., 2004). Using Bestkeeper Pfister and colleagues demonstrated CASC3 as one of the most stably expressed reference genes in diseased tissue (meningioma) but not in normal control tissue. The authors therefore do not recommend the use of CASC3 as a reference gene (Pfister et al., 2011). In the current study, RPL37A is the most unstable gene of all tested validated by geNorm. This finding was confirmed by NormFinder analysis being the third most unstably expressed of all genes tested which is mainly attributed to two expression level outliers in the OA group. Hence we do not recommend the use of RPL37A as a reference gene for normalization of OA-BMSC gene expression analyses. In a study validating candidate reference genes during differentiation of THP-1 monocytes into macrophages ACTB and RPL37A were identified as most reliable for qRT-PCR gene expression normalization (Maess et al., 2010). These findings are contradictory to those of the present study and demonstrate the importance of study design orientated reference gene evaluation. Usually the use of two and more different reference genes is recommended for gene expression normalization (Vandesompele et al., 2002; de Jonge et al., 2007). Notably Studer et al. suggest the single use of RPL13A as a reference gene with a high expression stability proven in different studies investigating mesodermal differentiation in BMSCs (Studer et al., 2012). As, in the present study, RPL13A was not covered by the commercially available assay used in this study we are not able to evaluate its role as a reference gene in the context of BMSCs and OA. In this study using the geNorm algorithm we were able to calculate a sufficient gene normalization using two most stably expressed reference genes. Therefore, we recommend using at least two of the most stably expressed reference genes for qRT-PCR based gene expression normalization in OA-BMSCs.
Conclusion
Contributions
Declaration of funding and role of funding source This work is part of the PhD thesis of TS funded by a European Social Fund (ESF) scholarship administered by the Sächsische Aufbaubank (SAB). The project was supported by the German Academic Exchange Service/Federal Ministry of Education and Research (D/09/04774).
Competing interests
Acknowledgment
Introduction Embryonic stem (ES) cells have the remarkable ability to replicate indefinitely while retaining the capacity to differentiate into functionally distinct cell types including neurons. The pluripotency and self-renewal of ES cells are regulated by a core transcriptional circuitry comprising of transcription factors, chromatin modulators, and small non-coding microRNAs (miRNAs) (Dejosez and Zwaka, 2012; Li et al., 2012; Pauli et al., 2011). The self-regulatory networks formed by transcription factors such as Oct4, Sox2, Nanog, Klf4, Esrrb, Tbx3, and Tcf3 regulate a wide range of downstream genes required for self-renewal and pluripotency (Boyer et al., 2005; Chambers and Tomlinson, 2009; Ivanova et al., 2006; Kim et al., 2008; Silva and Smith, 2008). In addition to the transcription factors, epigenetic changes associated with posttranslational modifications of histone proteins are known to regulate gene expression in ES cells (Li et al., 2012). Moreover, by directly interacting with ES cell transcription factors, miRNAs are shown to regulate the central signaling circuitry in ES cells (Pauli et al., 2011). The genetic deletion of key miRNA processing enzymes such as Dicer or Dgcr8 has been shown to impair cell cycle progression and cause defective differentiation (Murchison et al., 2005; Wang et al., 2008). Studies on the role of miRNAs in ES cells suggest that the regulatory function of the miRNA is often regulated through, and controlled by, various transcription factors important for self-renewal and pluripotency (Marson et al., 2008; Melton et al., 2010; Tay et al., 2008; Xu et al., 2009). An understanding of the interactions among these factors will be critical for the development of improved strategies to reprogram differentiated cells or induce differentiation of pluripotent ES cells to functionally distinct cell types.