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<h2>Replication Study: Transcriptional amplification in tumor cells with elevated c-Myc</h2> <p><br> <strong>Abstract:</strong></p> <p>As part of the <a href="https://osf.io/e81xl/wiki/home/" rel="nofollow">Reproducibility Project: Cancer Biology</a>, we published a Registered Report (<a href="https://elifesciences.org/articles/04024" rel="nofollow">Blum et al., 2015</a>), that described how we intended to replicate selected experiments from the paper "Transcriptional amplification in tumor cells with elevated c-Myc" (<a href="http://www.cell.com/cell/fulltext/S0092-8674(12)01057-4" rel="nofollow">Lin et al., 2012</a>). Here we report the results. We found overexpression of c-Myc increased total levels of RNA in P493-6 Burkitt’s lymphoma cells; however, while the effect was in the same direction as the original study (Figure 3E; <a href="http://www.cell.com/cell/fulltext/S0092-8674(12)01057-4" rel="nofollow">Lin et al., 2012</a>), statistical significance and the size of the effect varied between the original study and the two different lots of serum tested in this replication. Digital gene expression analysis for a set of genes was also performed on P493-6 cells before and after c-Myc overexpression. Transcripts from genes that were active before c-Myc induction increased in expression following c-Myc overexpression, similar to the original study (Figure 3F; <a href="http://www.cell.com/cell/fulltext/S0092-8674(12)01057-4" rel="nofollow">Lin et al., 2012</a>). Transcripts from genes that were silent before c-Myc induction also increased in expression following c-Myc overexpression, albeit less than active genes. Treating the data as paired, we found a statistically significant increase in gene expression for both active and silent genes upon c-Myc induction; however, the change in gene expression was found to be greater for active genes compared to silent genes. Finally, we report meta-analyses for each result, which suggests total RNA levels increased upon c-Myc induction, with increased gene expression observed in both active and, to a lesser extent, silent genes.</p> <hr> <h3>Contents</h3> <p><strong>Reports:</strong> Read the <a href="https://doi.org/10.7554/eLife.30274" rel="nofollow">Replication Study</a> or view the <a href="https://osf.io/n7tqh/" rel="nofollow">preprint versions</a>.</p> <p><strong>Note</strong>: In order to successfully run and knit the Replication Study Manuscript R Markdown file, you must install a series of necessary R packages. You can review the necessary packages included in the checkpoint manifest in the markdown file <a href="https://osf.io/vdrsh/" rel="nofollow">here</a> or run and knit the following r markdown file and they will be downloaded automatically.</p> <p>To reproduce the Replication Study manuscript text run this in <a href="https://www.rstudio.com" rel="nofollow">R Studio</a> (note: downloads <a href="https://osf.io/vdrsh/" rel="nofollow">R markdown file</a> directly from <a href="http://osf.io" rel="nofollow">osf.io</a>:</p> <pre class="highlight"><code>library(httr) library(pander) library(rmarkdown) GET(&quot;<a href="https://osf.io/vdrsh/?action=download" rel="nofollow">https://osf.io/vdrsh/?action=download</a>&quot;,write_disk(&quot;Replication_Study_48.Rmd&quot;, overwrite = T)) render(&quot;Replication_Study_48.Rmd&quot;, output_format = &quot;word_document&quot;)</code></pre> <p><br> Also, explore the Registered Report and materials related to the Registered Report <a href="https://elifesciences.org/articles/04024" rel="nofollow">here</a>.</p> <p><strong>Data and Material Availability:</strong> All associated data, protocols, analysis scripts, and other digital materials are available within this OSF project.</p> <p><strong>Experiments replicated</strong>: Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. <br> - <a href="https://osf.io/tfd57/wiki/home/" rel="nofollow">Western blot of c-Myc reactivation in tetracyclic-repressible system and total RNA expression during c-Myc re-activation</a> <br> -Replication of Figures 1B and 3E of Lin et al., 2012</p> <ul> <li><a href="https://osf.io/fn2y4/wiki/home/" rel="nofollow">Transcript levels during c-Myc re-activation and NanoString nCounter digital gene expression assay</a> <br> -Replication of Figure 3F and Table S1 of Lin et al., 2012</li> </ul> <p><strong>Meta-analysis</strong>: As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available <a href="https://osf.io/5yscz/wiki/home/" rel="nofollow">here</a>. <br></p> <p>Questions about the project can be directed to <a href="mailto:contact+rpcb@cos.io">contact+rpcb@cos.io.</a></p>
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