Response Issue #3: Validation of Results

[davebridges]

Regarding the experimental approach, it seems that the authors did not further validate the transcriptomic analysis by qPCR or western-blot, which drastically lessen the reliability of the results.

[davebridges]

To address the reproducibility of our findings, we have extensively compared our findings to published data both from single gene studies and from microarray studies on tissue culture cells. Neither of these are as broad in scope as our findings, but we have found good correlations between our findings and those of others in most cases, importantly for, IGF1, PTPN3, SOCS2, CISH, LPL and HSD11B1. In cases where we have not observed similar gene expression changes as proposed in the published literature we have discussed these as well, such as for PIK3R1. These descriptions provide solid validation for the reliability of our data.
We had considered performing qPCR studies to ‘re-validate’ some of our gene-expression findings but there is little evidence that qPCR analyses from the same samples will add any extra validity to our data so we decided to eschew those experiments. Ideally, we would re-validate our findings in a separate cohort of acromegalic patients, but due to the difficulty in accessing these samples, those experiments are not possible at this time.

[davebridges]

Updated Stock Answer

We had considered performing qPCR studies to ‘re-validate’ some of our gene-expression findings but there is little evidence that qPCR analyses from the same samples will add any extra utility to our data so we decided to eschew those experiments. Previous studies have shown extremely close correlations between qPCR and RNAseq data [1-4]. Ideally, we would re-validate our findings (potentially by qPCR) in a separate cohort of samples, but due to the difficulty in accessing these samples, those experiments are not possible at this time.

1. Griffith M, Griffith OL, Mwenifumbo J, Goya R, Morrissy a S, et al. (2010) Alternative expression analysis by RNA sequencing. Nat Methods 7: 843–847. doi:10.1038/nmeth.1503.
2. Asmann YW, Klee EW, Thompson EA, Perez E a, Middha S, et al. (2009) 3’ tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer. BMC Genomics 10: 531. doi:10.1186/1471-2164-10-531.
3. Wu AR, Neff NF, Kalisky T, Dalerba P, Treutlein B, et al. (2014) Quantitative assessment of single-cell RNA-sequencing methods. Nat Methods 11: 41–46. doi:10.1038/nmeth.2694.
4. Shi Y, He M (2014) Differential gene expression identified by RNA-Seq and qPCR in two sizes of pearl oyster (Pinctada fucata). Gene 538: 313–322. doi:10.1016/j.gene.2014.01.031.