Review for 10.1186/s12876-021-01707-7 (2021)

Reviewer’s report
Title: 
Gene Expression Profiling of Inflammatory Cytokines in Esophageal
Biopsies of Different Phenotypes of Gastroesophageal Reflux Disease: a cross-
sectional study
Version: 
2 
Date: 
11 Jan 2021
Reviewer's report:
PEER REVIEWER ASSESSMENTS: 
OBJECTIVE - Full research articles: is there a clear objective that addresses one
or several testable research questions? (Brief or other article types: is there a
clear objective?)
Yes - there is a clear objective
DESIGN - Is the current approach (including controls and analysis protocols)
appropriate for the objective?
No - there are minor issues
EXECUTION - Are the experiments and analyses performed with sufficient
technical rigor to allow confidence in the results?
Yes - experiments and analyses were performed appropriately
STATISTICS - Is the use of statistics in the manuscript appropriate?
Yes - appropriate statistical analyses have been used in the study
INTERPRETATION - Is the current interpretation/discussion of the results
reasonable and not overstated?
No - there are minor issues
OVERALL MANUSCRIPT POTENTIAL - Has the author addressed your
concerns sufficiently for you to now recommend the work as a technically sound
contribution? If not, can further revisions be made to make the work technically
sound?
Probably - with minor revisions
PEER REVIEWER COMMENTS: 
GENERAL COMMENTS: The manuscript has been rewritten for clarity and
correction, and the authors have tried to improve the content by attending to the
suggestions of editor and reviewers. However, there is still a lack of clarity in the
presentation of the qPCR data, failing to provide the data in a relevant manner.
REQUESTED REVISIONS: 
The authors have provided the requested data on cytokine levels in the new table
3. However, this is still difficult to interpret, and therefore I suggest the authors
to improve the title of the table and add units, so the readers understand directly
they are showing delta-delta-Ct values. In the graphs, they might need to clarify
they are showing just delta Cts, and define those two concepts so that readers not
familiar with qPCR can understand what the table and graphs show. Besides, the
method used is RT-qPCR, not just RT-PCR as mentioned somewhere in the
paper.
The graphs are still quite confusing, since they mostly show the same data
(contained already in figure 1) in different manners (else I fail to see the
difference). For instance, in figure 1 the relative levels of MMP3 for BE and
control are shown in a type of graph. The same data is repeated in figure 2, in
another graph with a different scale, in a different comparison (same Y axis,
different groups in X). The significance levels can be added to figure 1, or
summarized in Table 3 or other, if that was the point of all the other graphs. 
In the graphs, the Y axes basically report the ratio between the level of the gene
of interest and GAPDH. There are graphs where the authors show levels at 0.5%
the signal of the GAPDH, and even in figure 4b there are bars below 0.1% the
signal of the GAPDH. I wonder if the authors have revised the raw data and the
reactions were well adjusted. I have never seen such extremely low ratios
reported as a result of a qPCR analysis. I am not sure of the relevance of the
detection of the target genes at such extremely low Cts to yield such a low delta-
Ct.
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Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
No
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes