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P23 as loading control western blot8/19/2023 During development, these progenitors expand through cell proliferation, commit to distinct cell types and exit the cell cycle to generate either retinal neurons or the Müller glia in an evolutionary conserved birth order. Vertebrate retinas are composed of seven major cell types that are produced from multipotent progenitor cells. The retina is derived from the diencephalon, and is responsible for the conversion of electromagnetic energy into nerve impulses. In this study, we addressed this question focusing on retinal development. Considering the complexity of the neural tissue a major issue is the standardization of quantitative approaches to investigate expression patterns in response to specific treatments or throughout development. Concerning the study of the development of different tissues, these analyses can provide insights into complex regulatory networks that coordinate proliferation, cell commitment, differentiation and apoptosis. Gene expression analyses are crucial for the discovery and characterization of the roles for known genes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This study was funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), CNPq, and CAEN/International Society for Neurochemistry. Received: ApAccepted: JPublished: August 20, 2012Ĭopyright: © Rocha-Martins et al. PLoS ONE 7(8):įederal University of Rio de Janeiro, Brazil For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed.Ĭitation: Rocha-Martins M, Njaine B, Silveira MS (2012) Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. The stability of expression of seven putative reference genes ( Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. The paradoxical conclusion that anti-p41 IgM antibodies may be a sign of resistance to infection rather than a sign of infection should be given consideration.We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). Whether anti-p41 IgM antibodies are capable of inactivating infective spirochetes and thereby prevent infection in vivo is, however, not yet clarified. We observed that six out of nine sera with such antibodies could immobilize a B. Based on our data we hypothesize that up to 1.5% of the population may have natural IgM antibodies against p41 in their sera. Five blood donor sera and five cord sera showed an IgM reactivity against p41. Sera from 330 blood donors and 72 cord sera were also screened for anti-p41 IgM. Two whole cell enzyme-linked immunosorbent assay (ELISAs), two immunofluorescence assays and Western blotting were not useful as confirmatory tests. The remaining 48 patients were negative in this test and were considered as false-positives. A confirmatory IgM Western blot gave a positive reaction in 5 patients out of 53 patients with little or no clinical evidence of B. Ten of the 63 patients recently had symptoms of erythema migrans. Sixty-three patients had repetitive positive tests for IgM anti-p41 antibodies and negative tests for anti-p41 IgG antibodies. During a 1-year interval 2403 patients were referred to our department for B. We performed a prospective study to investigate the biological significance and diagnostic specificity of anti-p41 immunoglobulin (Ig)M antibodies against Borrelia burgdorferi.
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