Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes
and Ricardo Fernández-de-Misa 1,2,5,*,†
Round 1
Reviewer 1 Report
The authors evaluated reference genes for RT-qPCR of FFPE cSCC and MM samples. Based on published studies, they selected 4 genes and performed the study on 3 of them. The experiments are appropriate and adequately conducted. The results will be useful for studies of retrospective cohorts of cSCC and MM, for which FFPE blocks are available.
Author Response
RESPONSE TO REVIEWER 1:
We are very pleased that you find this work valuable and useful for other studies in field.
English language and style were checked, and minor spell mistakes were corrected as required.
Elizabeth Córdoba Lanús Ph.D.
Ricardo Fernandez-de-Misa-Cabrera Ph.D.
Author Response File: 
Author Response.docx
Reviewer 2 Report
García-Pérez et al. examined stability of ACTB, TFRC, HPRT1, and TBP mRNAs to identify suitable internal controls for normalization of gene expression when FFPE MM and CSCC samples are used. They showed that ACTB, TFRC, and HPRT1 can be stably detected by real-time qPCR using cDNA reverse-transcribed from mRNA. Therefore, they concluded that one or two of these mRNAs may be suitable as internal controls for normalization of target gene expression. Their results might be informative, but before consideration of publication, the authors should respond to comments described below.
1) Lack of technical information. It is not clear which regions were amplified by qPCR. For example, primer sequences, amplicon length, GC contents of target regions should be summarized in a Table. Those kinds of information would be necessary to compare efficiency of DNA amplification.
2) The authors used commercial kits of primers/probes for evaluation of cDNA amplification. Is it essential to use those commercial products? Can researchers use self-designed primers targeting other sequences in the same genes?
3) Conclusions were different according to software. What is the best internal control in the end? Which software should be used? Those kinds of information would be important for readers.
4) The authors recommended combinatorial use of two internal controls for normalization. However, it is not clear how two (e.g., ACTB and HPRT1) are used in combination for normalization and interpretation of gene expression. A typical example would be useful for readers.
5) The authors examined just stability of potential internal control mRNAs in this study. It would be useful to actually examine a target gene expression using those potential internal controls.
6) Page 5, the first paragraph: I could not find a figure on the description “Using NormFinder software, the best combination of two reference genes for normalizing CSCC tissue were TFRC and HPRT1 (stability value 0.079) and for MM HPRT1 and ACTB resulted the most stable combination (0.14).”
Author Response
RESPONSE TO REVIEWER 2
García-Pérez et al. examined stability of ACTB, TFRC, HPRT1, and TBP mRNAs to identify suitable internal controls for normalization of gene expression when FFPE MM and CSCC samples are used. They showed that ACTB, TFRC, and HPRT1 can be stably detected by real-time qPCR using cDNA reverse-transcribed from mRNA. Therefore, they concluded that one or two of these mRNAs may be suitable as internal controls for normalization of target gene expression. Their results might be informative, but before consideration of publication, the authors should respond to comments described below.
We appreciate that you find this work interesting and noteworthy. We have tried to clarify every one of your doubts. Also, we included sentences that reflect these clarifications in the new version of the manuscript.
Point 1: Lack of technical information. It is not clear which regions were amplified by qPCR. For example, primer sequences, amplicon length, GC contents of target regions should be summarized in a Table. Those kinds of information would be necessary to compare efficiency of DNA amplification.
Thank you for the valuable comment. As suggested a table with all the information related to target reference genes and reference of the commercial assays that contain the primers/probes used in the experiments was elaborated and referenced in the methods proper section and named as “Table 2” (Candidate genes and Reference genes expression by qPCR sections: Materials and Methods)
Point 2: The authors used commercial kits of primers/probes for evaluation of cDNA amplification. Is it essential to use those commercial products? Can researchers use self-designed primers targeting other sequences in the same genes?
In this type of studies, commercial assays with TaqMan probes are commonly used due to their high reliability. These kinds of commercial products are already tested and referenced by other author’s research. Of course, primers of our own design could be used to target the desired sequences of the same genes, although it is more robust to use the customized TaqMan probes since they have a high sensitivity and specificity.
Point 3: Conclusions were different according to software. What is the best internal control in the end? Which software should be used? Those kinds of information would be important for readers.
The use of the three methods is important because they provide us complementary information increasing the robustness of our in-silico analysis and in consequence the reliability in our findings. This has already been reflected in the discussion section (paragraph 5 of the discussion) where we stated that: “Combined use of these methods together with BestKeeper algorithm allowed us to show which of these genes is the most stable and which is the best combination between them for CSCC and MM FFPE samples gene expression studies”. And we concluded that: We suggest that ACTB and HPRT1 in combination are the appropriate reference genes for the normalization of gene expression studies in MM FFPE samples, as is the combination of TFRC and HPRT1 reference genes in CSCC FFPE tissue.
It is not recommended to use just a single software for this kind of analysis. In this case, NormFinder is the more complete software, because it allows us to obtain variability data of a solely gene or by combining the best two resulting genes. In this sense, implementing these combined reference genes in expression studies may provide higher stability values when compared to the use of a single gene. However, its highly recommended to test expression data using at least two or three methods, as we did in this study.
Following reviewer suggestion, we added a paragraph with this recommendation to scientific readers in the discussion section (paragraph 5).
Point 4: The authors recommended combinatorial use of two internal controls for normalization. However, it is not clear how two (e.g., ACTB and HPRT1) are used in combination for normalization and interpretation of gene expression. A typical example would be useful for readers.
For using the resulting 2 genes in combination for the normalization of gene expression studies an average of the Ct values of the 2 reference genes must be calculated for each sample. This resulting Ct value will be used for correcting the target gene Ct value of each sample, while using the calculations required in the normalization method chosen (for example: 2-△△Ct). As suggested, this procedure was added and explained in the results section in relation to the finding of the combined 2 genes as the best option for normalization of CSCC and MM tissues (Results section, paragraph 3: Expression stability of the candidate reference gene)
Point 5: The authors examined just stability of potential internal control mRNAs in this study. It would be useful to actually examine a target gene expression using those potential internal controls.
The aim of the present study was to elucidate which is the most appropriate reference gene suitable for gene expression studies in FFPE samples of CSCC and MM, since confident data about them was scarce in the literature. This study represents the kind of research that must be performed prior for initiating genetic expression studies on genes of interest. Every study might be experimentally validated for specific tissues or cell types and specific experimental designs.
Point 6: Page 5, the first paragraph: I could not find a figure on the description “Using NormFinder software, the best combination of two reference genes for normalizing CSCC tissue were TFRC and HPRT1 (stability value 0.079) and for MM HPRT1 and ACTB resulted the most stable combination (0.14).”
Thank you for the observation. These data were added to the Figure 3A and cited in the text in the Results section in relation with the above findings (Results section, paragraph 3: Expression stability of the candidate reference gene)
We hope that this response and all the improvements we have made is worthy of your approval.
Elizabeth Córdoba Lanús Ph.D.
Ricardo Fernandez-de-Misa-Cabrera Ph.D.
Author Response File: Author Response.docx
