Author's response to reviews Title: Validation of putative housekeeping genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR. Authors:

The abstract provides a good overview of the findings. The authors are correct that reference genes are often referred to as house keeping genes. This is actually a misleading practice. While reference genes can be house keeping genes, not all house keeping genes are suitable reference genes and not all reference genes need to be house keeping genes. The authors should stick to the term reference genes throughout this manuscript and avoid using house keeping genes apart from an initial mention in the background. We thank the reviewer for this comment and in the revised version of our manuscript we kept the term “reference genes” throughout this manuscript with exception of an initial mention of “house keeping genes” in the background. Background: This provides a comprehensive overview but is generally too long. Cut down by at least 1⁄4 and structure with paragraphs. As suggested by the reviewer, the background was cut down by approximately 1/3 (32 lines currently compared to 51 lines in the original version) and structured with paragraphs. Page 3 sentence “An ideal HKG should present stable expression....” See HKG point made by abstract. Also what the authors are alluding to by this sentence is generally correct, however what do they mean by “stable”? It would be more accurate to state that an ideal reference gene needs to be unaffected by the experimental conditions while also having low variation in gene expression. Their next sentence “Otherwise, the noise....” is incorrect, as the noise (error) of the assay is not capable alone of generating erroneous results. For this to occur the reference gene would need to be affected by the experiment (causing directional shift or statistical bias). We agree that the interpretation of the reviewer about ideal HKG is more accurate than ours, and have corrected it accordingly (please see page 4, lines 14 15) and deleted the sentence “Otherwise, the noise....”. Sentence starting “furthermore, conventional normalisation strategies.....”. This reviewer feels that this should be omitted as it is misleading. Erroneous normalisation is caused by poor validation. While multiple reference genes may reduce the chances of this, if the wrong genes are chosen then they can also bias the results. Multiple reference genes facilitate much finer measurements than a single reference gene, as the trends that occur due to experimental error can be observed and compensated for. We agree with the reviewer’s opinion about the sentence “furthermore, conventional normalisation strategies.....”, and have omitted it.