Review for 10.1186/1471-230X-14-1 (2014)

Reviewer's report
Title:Over-expression of COX-2 mRNA in colorectal cancer
Version: 1Date:15 July 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
RT-PCR. Expression data was compared using three different normalization
strategies- tissue weight and two individual housekeeping genes (B2M and
GAPDH).
Their main finding is that PTGS2 mRNA is higher in tumor than normal colorectal
mucosa, an observation that is not particularly novel. They also observed that the
extent to which PTGS2 mRNA is upregulated in tumor over normal tissue
depends on the standardization method used, also a known phenomenon.
Problems with using individual housekeeping genes, in particular GAPDH, are
well known, as the author notes in the Discussion. While using tissue weight
could be an attractive alternative normalization strategy in some situations, the
data presented are inadequate to reach the conclusion that this method is
somehow superior.
Major Compulsory Revisions
1. The authors argue that tissue weight is a superior choice over individual
housekeeping genes as a normalization strategy. This appears to be based on
the fact that this normalization method yielded the highest percentage of tumors
with upregulated PTGS2 expression, rather than a careful comparison of the
precision and accuracy of the different normalization strategies. Without such
data, it is impossible to say whether the tissue weight is a superior normalization
strategy and thus such a conclusion is not supported by the data offered.
2. The authors also argue that tissue weight is a simpler normalization strategy
compared to B2M or GAPDH, due to the variable expression of these two genes.
However, the authors do not provide any information on the variability of the
weight measurements and how that may impact their findings.
3. No true replicate samples were analyzed, and no %CV was reported for any
assay. Thus the accuracy and precision of the measurements generated are not
known as the error associated with the weighing procedure, the
reverse-transcriptase procedure, and inter-assay variability are not captured for
any method.
4. The author only looked at two housekeeping genes, and did not look at the
combination of multiple reference genes, using algorithms such as geNorm
(http://medgen.ugent.be/~jvdesomp/genorm/).
5. One alternative to using housekeeping genes that has been suggested in the
literature is to measure cDNA input through the use of fluorescein-labeled
oligonucleotides such as OliGreen (Invitrogen), and use that value as a
normalizer (Rhinn H, Scherman D, Escriou V (2008) One-step quantification of
single-stranded DNA in the presence of RNA using Oligreen in a real-time
polymerase chain reaction thermocycler. Anal Biochem 372: 116–118.) This
would have been a useful and potentially more objective measurement.
Minor Essential Revisions
1. No discussion in the methods section of the type of balance used to weigh the
tissues, or mention of the accuracy/precision of such weight measurements is
included.
2. No discussion of the limitation of tissue weight as a normalization strategy are
presented. For instance, this is not a useful strategy for formalin fixed tissue, and
thus irrelevant for large epidemiological studies using clinical blocks.
3. The authors should use the accepted HUGO gene symbol- PTGS2 when they
are discussing mRNA expression of COX-2.
http://www.genenames.org/data/hgnc_data.php?hgnc_id=9605
Level of interest: An article of limited interest
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I have no competing interests