Isolation of Chromatophores from Brown Trout (Salmo trutta) Skin
Round 1
Reviewer 1 Report
Dear authors,
I read thoroughly your manuscript “Isolation of Chromatophores from Brown Trout (Salmo trutta) 2 Skin” and I am pleased by the high scientific soundness of your work. The method you tested has simplified the isolation of single cells from salmonoid skin.
I would have only to ask to add to the manuscript how many samples did you used overall (Line 71) and in each of the three sampling approaches.
In Lines 235-241, you state that the optimal time for digestion in the collagenase solution was 40 to 50 minutes, but in Table 1 I see the 60 minutes as the optimal time. Please check this discrepancy.
How does the paragraph of Lines 339-344 add value to the manuscript? If you think it is somehow irrelevant, then it may be omitted.
Last, in Line 348, where you present the Final Protocol, you may rename it as SOP (Standard Operating Procedure) and add same introductive info for the protocol, such as Aim of the SOP, References, Terminology used, Basic methodology, Materials needed, Equipment needed, Precautions, and after these you continue with the Steps of the actual protocol.
Author Response
Response to Reviewer 1 Comments
Dear authors,
I read thoroughly your manuscript “Isolation of Chromatophores from Brown Trout (Salmo trutta) 2 Skin” and I am pleased by the high scientific soundness of your work. The method you tested has simplified the isolation of single cells from salmonoid skin.
Point 1: I would have only to ask to add to the manuscript how many samples did you used overall (Line 71) and in each of the three sampling approaches.
Response 1: The information about the samples used was added, please see Line 79.
Point 2: In Lines 235-241, you state that the optimal time for digestion in the collagenase solution was 40 to 50 minutes, but in Table 1 I see the 60 minutes as the optimal time. Please check this discrepancy.
Response 2: Thank you to draw attention to this mistake. The numbers were corrected to show the right and same result in text and Table 1.
Point 3: How does the paragraph of Lines 339-344 add value to the manuscript? If you think it is somehow irrelevant, then it may be omitted.
Response 3: We think that these results could be relevant in terms of isolated cell viability proof, but we moved the information to conclusion where it was incorporated in the changed text and put in more context.
Point 4: Last, in Line 348, where you present the Final Protocol, you may rename it as SOP (Standard Operating Procedure) and add same introductive info for the protocol, such as Aim of the SOP, References, Terminology used, Basic methodology, Materials needed, Equipment needed, Precautions, and after these you continue with the Steps of the actual protocol.
Response 4: We added some information to the final protocol as suggested by the reviewer.
Reviewer 2 Report
In the current manuscript, the authors describe a protocol for the isolation of chromatophores from brown trout skin. They collected skin biopsies from red and black spots, digested the tissues with trypsin and or collagenase at different concentrations and incubation time, selected the cells, and measured the expression of nine genes that were previously reported to be upregulated in red-spotted skin regions in brown trout. The idea itself is interesting, however the study has several major flaws which reduces the confidence in the results and the protocol.
- The aim of developing this technique is for single cell analysis. In single cell RNA-Seq, almost all gene transcripts are quantified. These genes are expected to have variable expression levels. For example, some transcripts will be abundant while others will be rare. The current technique developed by the authors does not address this point. The authors rely only on measuring the expression of genes that are upregulated. How about the expression other genes?
- Why did authors use only one biological replicate for gene expression analysis?
- The authors pooled 15-20 cells for gene expression analysis, why?? This technique is intended for single cell analysis; therefore, gene expression should be measured in single cells not cell pools.
- The authors did not use a valid control for gene expression analysis. The tissue samples were exposed to conditions that are expected to affect the RNA quality and quantity. Therefore, the expression levels measured in these samples are lower than the actual levels in the untreated fish skin due to RNA degraded by the isolation technique. The question is: how low are these levels? The good technique should preserve as much as possible of the RNA. Therefore, the authors should have used a normal untreated sample from the fish spotted skin to compare the expression in the isolated cells and assess the effects of their technique on the RNA. Another approach is to use a housekeeping gene that is expressed evenly across tissues and measure its expression in the isolated cells and compare it to single cells from the fish blood.
Author Response
Response to Reviewer 2 Comments
In the current manuscript, the authors describe a protocol for the isolation of chromatophores from brown trout skin. They collected skin biopsies from red and black spots, digested the tissues with trypsin and or collagenase at different concentrations and incubation time, selected the cells, and measured the expression of nine genes that were previously reported to be upregulated in red-spotted skin regions in brown trout. The idea itself is interesting, however the study has several major flaws which reduces the confidence in the results and the protocol.
Point 1: The aim of developing this technique is for single cell analysis. In single cell RNA-Seq, almost all gene transcripts are quantified. These genes are expected to have variable expression levels. For example, some transcripts will be abundant while others will be rare. The current technique developed by the authors does not address this point. The authors rely only on measuring the expression of genes that are upregulated. How about the expression other genes?
Response 1: We are aware of the fact that gene expression is variable and also different when comparing different cell types. It is true that the tested genes were chosen among upregulated genes in red spotted skin region of brown trout, where subtype 2 erythrophores prevail among chromatophore cells. On the other hand, these genes were downregulated in black spotted skin region, where melanophores (also analysed in this study) are the main chromatophore cell present. When extrapolating these results to one cell type, we expected that tested genes would be upregulated in subtype 2 erythrophores and downregulated in melanophores, while expression in subtype 1 erythrophores, present in black spots beside melanophores, was not known and could not be predicted from previous results, where the whole skin was used for RNA isolation. This way, we in fact analysed also genes that were expected to be expressed at very low amount or not expressed at all in melanophores and subtype 1 erythrophores. As written in the text, the expression analyses were used to check the specificity of the isolation protocol, and since the genes were somehow selected from previous analyses, they do not coincide with or indicate the overall expression profile of isolated cells, that could be only determined with downstream application of single cell RNA-seq. Single cell RNA-seq was not performed within this study, but is mentioned as one of possible downstream applications using single cell suspension. We have changed the conclusion completely, and added the term preparation of single cell suspension beside single cell isolation to more precisely describe presented protocol. In addition, with changed text in conclusion, we believe we have reduced the focus of the manuscript to single-cell RNA-seq.
Point 2: Why did authors use only one biological replicate for gene expression analysis?
Response 2: The focus of the manuscript was not meant to be on expression analyses but rather on protocol for single-cell solution preparation and single-cell isolation. Gene expression was included in the manuscript as one of possible way to show that the optimised protocol results in successful selection and isolation of cells of particular cell type. We believe that also with one biological sample we managed to show differential expression of selected genes in cells of different cell type. We agree with the reviewer that a meaningful conclusion could not be drawn from one biological replicate. Therefore, we have reduced the paragraph describing the expression profile, and focused the text more on differential expression, but not to expression of a particular gene.
Point 3: The authors pooled 15-20 cells for gene expression analysis, why?? This technique is intended for single cell analysis; therefore, gene expression should be measured in single cells not cell pools.
Response 3: The SYBR Green Fast Advanced Cells-to-CT Kit is designed for 10–100,000 cultured cells/sample. To the best of our effort, we haven’t found a kit to be able to isolate the RNA from a single cell and use this isolate for standard qPCR analyses. Considering the range of the cell number recommended by the kit supplier, we approached the lowest recommended number of cells used for RNA isolation. All the cells in one isolate were of the same chromatophore type, therefore, we expect that they share the expression profile. We have to point to the fact that even for single cell RNA-seq or Droplet Digital™ PCR, where expression of a single cell is analysed, the starting material is represented by a population of isolated individual cells (single-cell suspension), and not by single cell per se.
Point 4: The authors did not use a valid control for gene expression analysis. The tissue samples were exposed to conditions that are expected to affect the RNA quality and quantity. Therefore, the expression levels measured in these samples are lower than the actual levels in the untreated fish skin due to RNA degraded by the isolation technique. The question is: how low are these levels? The good technique should preserve as much as possible of the RNA. Therefore, the authors should have used a normal untreated sample from the fish spotted skin to compare the expression in the isolated cells and assess the effects of their technique on the RNA. Another approach is to use a housekeeping gene that is expressed evenly across tissues and measure its expression in the isolated cells and compare it to single cells from the fish blood.
Response 4: Thank you for this comment. Indeed, we haven’t included a control demonstrating the potential harmful effects of isolation procedure to the cell and their expression levels. It is almost impossible to compare the expression of whole untreated tissue sample to expression of 15-20 cells. The other approach mentioned was applied and is mentioned in the text (L. 398). We used the same protocol to collect blood cells as was used for selection and isolation of chromatophores (mouth pipette; paragraph 3.4). RNA was isolated from collected blood cells and expression of housekeeping gene rps20 tested using SYBR™ Green Fast Advanced Cells-to-CT™ Kit. The results of this expression analyses confirm the appropriateness of presented protocol, since the expression level was comparable (please, see L. 398 – 403).
Round 2
Reviewer 2 Report
I would like to thank the authors for diligently revising their manuscript, response to the reviewer's comments, and clarifying some of the points that were not clear. The manuscript is in better shape now.
However, I would highly recommend that the conclusions of the study be shortened and concise. This information can be moved to the discussion section. Another comment is that the limitations of the study should be clarified and mentioned at a suitable paragraph (for example, at the end of the discussion and before the conclusion). This will guide future research to address these limitations including the effects of cell handling on the RNA quality and quantity, especially if this protocol is to be used for any gene expression studies.
Author Response
I would like to thank the authors for diligently revising their manuscript, response to the reviewer's comments, and clarifying some of the points that were not clear. The manuscript is in better shape now.
However, I would highly recommend that the conclusions of the study be shortened and concise. This information can be moved to the discussion section. Another comment is that the limitations of the study should be clarified and mentioned at a suitable paragraph (for example, at the end of the discussion and before the conclusion). This will guide future research to address these limitations including the effects of cell handling on the RNA quality and quantity, especially if this protocol is to be used for any gene expression studies.
Reply: As suggested by the reviewer, the conclusion was shortened and most of the previous text in this section moved to the appropriate position within Methods, Results and Discussion. In addition, besides applications also some potential limitations of the presented protocol were mentioned and discussed. All the changes made to the text are visible with track changes option on.
Author Response File: 
Author Response.docx
