Reviewer's report
Title:Over-expression of COX-2 mRNA in colorectal cancer
Version:2Date:7 November 2013
Reviewer: Karen W. Makar
Reviewer's report:
The authors present a study in which they examined the expression of PTGS2
(the gene encoding the Cox-2 enzyme) in 60 tumor and normal tissue pairs via
real-time RT-PCR. Expression data was compared using three different
normalization strategies- tissue weight and two individual housekeeping genes
(B2M and GAPDH). While using tissue weight could be an attractive alternative
normalization strategy, the data presented are still inadequate to reach the
conclusion that this method is superior.
Major Compulsory Revisions:
1. The conclusion that the tissue normalization strategy is “more accurate” is still
unfounded. This paper does not present experimental data on inter- and intra-
assay variance, or biological or technical replicates. This data and a statistical
comparison of the accuracy and/or precision of the tissue normalization vs.
housekeeping gene methods would be necessary to support such a conclusion.
Either the statements about superior accuracy need to be curtailed in recognition
of the absence of rigorous methodological data, or proper assay development
experimental data need to be included in tables/figures to support these
statements. The tissue normalization procedure has promise, but the authors
should not overstate what their data supports.
2. Please provide details (e.g. formula) explaining the tissue normalization
strategy in the Methods. The authors state that for each sample 1ug of RNA was
converted into cDNA. Given that larger tissues would likely yield more RNA (and
the RNA yields and tissue weights are not provided), it is unclear from the text
how the ug RNA yield/mg tissue per sample is incorporated in the normalization
procedure.
Minor revisions-
Tissue weight normalization alone would not capture variability in RNA quality,
which is an essential component of the MIQE guidelines (ref 24) as discussed
recently in Bustin et al, Nature Methods Oct 2013. This is not discussed.
Background paragraph 3, 3rd sentence: there is a large space between
“subjected to” and “was suppressed” and this sentence makes no sense.
Discussion paragraph 7, missing a period after “tumor tissue.”
Level of interest: An article of limited interest
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no competing interests
Reviewer's report
Title:Over-expression of COX-2 mRNA in colorectal cancer
Version:2Date:7 November 2013
Reviewer: Karen W. Makar
Reviewer's report:
The authors present a study in which they examined the expression of PTGS2
(the gene encoding the Cox-2 enzyme) in 60 tumor and normal tissue pairs via
real-time RT-PCR. Expression data was compared using three different
normalization strategies- tissue weight and two individual housekeeping genes
(B2M and GAPDH). While using tissue weight could be an attractive alternative
normalization strategy, the data presented are still inadequate to reach the
conclusion that this method is superior.
Major Compulsory Revisions:
1. The conclusion that the tissue normalization strategy is “more accurate” is still
unfounded. This paper does not present experimental data on inter- and intra-
assay variance, or biological or technical replicates. This data and a statistical
comparison of the accuracy and/or precision of the tissue normalization vs.
housekeeping gene methods would be necessary to support such a conclusion.
Either the statements about superior accuracy need to be curtailed in recognition
of the absence of rigorous methodological data, or proper assay development
experimental data need to be included in tables/figures to support these
statements. The tissue normalization procedure has promise, but the authors
should not overstate what their data supports.
2. Please provide details (e.g. formula) explaining the tissue normalization
strategy in the Methods. The authors state that for each sample 1ug of RNA was
converted into cDNA. Given that larger tissues would likely yield more RNA (and
the RNA yields and tissue weights are not provided), it is unclear from the text
how the ug RNA yield/mg tissue per sample is incorporated in the normalization
procedure.
Minor revisions-
Tissue weight normalization alone would not capture variability in RNA quality,
which is an essential component of the MIQE guidelines (ref 24) as discussed
recently in Bustin et al, Nature Methods Oct 2013. This is not discussed.
Background paragraph 3, 3rd sentence: there is a large space between
“subjected to” and “was suppressed” and this sentence makes no sense.
Discussion paragraph 7, missing a period after “tumor tissue.”
Level of interest: An article of limited interest
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no competing interests
Please review this paper.
user
Reviewer's report
Title:Over-expression of COX-2 mRNA in colorectal cancer
Version:2Date:7 November 2013
Reviewer: Karen W. Makar
Reviewer's report:
The authors present a study in which they examined the expression of PTGS2
(the gene encoding the Cox-2 enzyme) in 60 tumor and normal tissue pairs via
real-time RT-PCR. Expression data was compared using three different
normalization strategies- tissue weight and two individual housekeeping genes
(B2M and GAPDH). While using tissue weight could be an attractive alternative
normalization strategy, the data presented are still inadequate to reach the
conclusion that this method is superior.
Major Compulsory Revisions:
1. The conclusion that the tissue normalization strategy is “more accurate” is still
unfounded. This paper does not present experimental data on inter- and intra-
assay variance, or biological or technical replicates. This data and a statistical
comparison of the accuracy and/or precision of the tissue normalization vs.
housekeeping gene methods would be necessary to support such a conclusion.
Either the statements about superior accuracy need to be curtailed in recognition
of the absence of rigorous methodological data, or proper assay development
experimental data need to be included in tables/figures to support these
statements. The tissue normalization procedure has promise, but the authors
should not overstate what their data supports.
2. Please provide details (e.g. formula) explaining the tissue normalization
strategy in the Methods. The authors state that for each sample 1ug of RNA was
converted into cDNA. Given that larger tissues would likely yield more RNA (and
the RNA yields and tissue weights are not provided), it is unclear from the text
how the ug RNA yield/mg tissue per sample is incorporated in the normalization
procedure.
Minor revisions-
Tissue weight normalization alone would not capture variability in RNA quality,
which is an essential component of the MIQE guidelines (ref 24) as discussed
recently in Bustin et al, Nature Methods Oct 2013. This is not discussed.
Background paragraph 3, 3rd sentence: there is a large space between
“subjected to” and “was suppressed” and this sentence makes no sense.
Discussion paragraph 7, missing a period after “tumor tissue.”
Level of interest: An article of limited interest
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no competing interests
assistant