PONE-D-21-39912Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenopharyngodon idella)PLOS ONE

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Additional Editor Comments:

All data and its representation including raw sequencing data (NCBI) should be accessible for the reviewers.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In their manuscript “Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenopharyngodon idella)” Yinli et al. studied miRNA transcriptome and miRNA–mRNA interaction networks in grass carp normal spleen tissue. Specifically, they show:

• Identified 324 known conserved miRNAs and 9 novel miRNAs by using bioinformatics,

• 23 families of miRNAs showed highly conservation between vertebrates and invertebrates and 14 of them are present only in fish,

• Expression patterns of miRNAs consistent between one-year-old and three-year-old spleen.

This study is descriptive and provides resources for understanding the molecular details of the spleen development in the grass carp.

The presented conclusions are well supported by the data. Appropriate controls have been used to support the experiments.

Reviewer #2: In this manuscript Zhao et.al. report expression of sRNAs in the spleen of grass carp (Ctenopharyngodon idella). Using the sRNA expression data, the authors have identified conserved as well as unique microRNA in young and aged grass carp spleen and predicted potential targets and interaction networks. This report is potentially important considering the importance of microRNAs in the regulation of fish immunity and spleen function. However, the data are not fully accessible/legible in order to make evaluation of the manuscript.

1. Please rectify the raw data archive accession number. The information is submitted which says, “The raw sequencing data had been deposited into the NCBI database Sequence Read Archive under number PRJNA328412 (mRNA) and PRJNA645243 (miRNA)”. However, no entry was found using accession number PRJNA645243. Inaccessibility of the data may impede the conclusions of the paper.

2. Figures are illegible, specifically Figures 1,3,5 and 6 are inaccessible to make any assessment of the data presented. Other figures also need significant improvement in presentations.

Minor

1. Figure 4: Please provide the qPCR primer sequences or catalog number if commercially available.

Line 17 and 99: Six versus two library construction were respectively mentioned. Authors needs to clarify the differences.

Reviewer #3: The spleen is found in nearly all vertebrates and is one of the major lymphoid organs. It primarily acts as a blood filter thereby recognizing and removing old or damaged red blood cells, and also plays important role in regard to the immune system. Previously, microRNAs are known to play a vital role in regulating the immune function of the spleen, although their expression and interaction networks are still poorly understood in late post-natal development.

The study “Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenophariyngodon idella)” by Zhao et al. reported for the first time the identification of the miRNA transcriptome and also provided the miRNA-mRNA interaction networks. They have constructed small RNA libraries from spleen tissues of one and three year old grass carp, and with the help of bioinformatic analysis have identified a total of 324 known conserved miRNAs and 9 novel miRNAs. The authors showed that 23 families were highly conserved between vertebrates and invertebrates while 14 others were present only in fish, and miRNA expression patterns were highly consistent between the two spleen groups. The data obtained have been expanded to the number of known miRNAs, thus providing important information to better understand spleen development of grass carp at the molecular level.

The idea and study designed is well conceived and well performed, and the findings in the manuscript are very interesting. The data and results are well presented and very convincing. Overall, the authors have done a wonderful job, however, there minor points that need to be addressed prior publication.

1. The main conclusion from the abstract and the introduction stresses upon the novel finding of the miRNA transcriptome and miRNA-mRNA interaction network, whereas in the discussion section the conclusion was stressed upon miRNA regulation of the lipid metabolism and spleen function. Kindly rearrange such that a common conclusion is presented throughout the manuscript.

2. In Line 99, it says “two miRNA libraries were constructed from the spleen tissue of one year old and three year old”, whereas in line 140 it says “ six miRNA libraries were constructed from spleen tissue of grass carp at one year and three years of age”. So what is the exact number of libraries being constructed?

3. The resolution of the figures need further enhancement, as it is difficult to visualize and read.

4. Although, it is mentioned that RNA isolation from spleen tissues was carried out using Trizol. But what type of method was used to homogenize the spleen tissues?

5. What is the amount of total RNA used to make each miRNA library construction by TruSeq Small RNA Sample prep Kit for sequencing purposes?

6. For qRT-PCR method section, kindly provide a brief description on how the cDNA was made? Also please include a reference of the method used for calculation of the miRNA expression level.

7. Kindly maintain a uniformity of the numbering for figures including legends and text in the results section. For instance, Figure 1A is presented differently in the figure legends as (a) instead of (A), and the same is for figure 3, 4, and 6.

8. The subfigure number A, B, C,… and F should be also mentioned in the legend for figure 4 along with the respective miRNA.

9. The figure legends for figures 7, 8 and 9, should come after the result description, but not before as presented in the manuscript.

10. Lines 380, 391 and 400, the sentences start with the same words “ In the miRNA-mRNA interaction networks associated with”, kindly reframe each of the sentences differently.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenopharyngodon idella)

PONE-D-21-39912R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: For Fig 3 and 4, even though authors did triplicate, but didn't show error bars. Please check and correct it.

Reviewer #2: Authors have addressed all my comments, made data and figures legible. The data support the conclusions of the manuscript.

Reviewer #3: Plos One Reports-PONE-D-21-39912R1:

In their manuscript “Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenophariyngodon idella)”, Zhao et al. studied on miRNA transcriptome and the miRNA-mRNA interaction networks. This detailed study and data obtained do provides important resources at the molecular levels in understanding how spleen develops in grass carp.

The authors have done a wonderful job in accommodating all the comments that were suggested and I am very pleased to recommend the present version of the manuscript for publication in this journal.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No