Identification of suitable reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) in the fibrotic phase of the bleomycin mouse model of pulmonary fibrosis

Oula Norman; Jarkko Koivunen; Joni M. Mäki; Taina Pihlajaniemi; Anne Heikkinen

doi:10.1371/journal.pone.0276215

Peer Review History

Original SubmissionAugust 12, 2022
6 Sep 2022 Decision Letter - Kyung-Wan Baek, Editor PONE-D-22-22612Identification of suitable reference genes for normalization of reverse transcription quantitative real-time PCR in the fibrotic phase of the bleomycin mouse model of pulmonary fibrosisPLOS ONE Dear Dr. Heikkinen, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Oct 21 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Kyung-Wan Baek, Ph.D. Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments : Your manuscript will be considered suitable for publication in PLos ONE if it undergoes appropriate revisions. Please revise the manuscript to reflect the reviewers' comments. [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: Yes ****** 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 ****** 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: This paper presents proper reference genes for the RT-qPCR in the pulmonary fibrosis mouse model after treatment of the Bleomycin. In the fibrosis mouse model, the reference gene selection is an important and appropriate study for verifying gene expression. If the authors solve the minor issues below, this paper will be published to the PLoS One. Manuscript Overall: Gene Symbol should be used as an italic, and all capital letters. Line 2: Please provide abbreviation of reverse transcription quantitative real-time PCR as (RT-qPCR) in title. Line 17: two separate algorithms → Please present specific names. Line 85-86: “ab libitum” please provide as italic. Line 130 Table 1, 5th and 7th columns: please provide direction of primer sequences (5’-3’). Figure 3 and 5: There may be confusion in sample names. “Untreated” → “Untreated samples”, “Bleomycin” → “Bleomycin-treated samples”. If you lack space, you can use abbreviations. "UC" and "BTS". The difference between the groups according to the shape of each point (round point, square point) should be described in each figure legend. Reviewer #2: The ms. by Norman et al. presents data regarding a number of genes that could serve as reference genes in the bleomycin mouse model. These are proposed as stable denominators for gene expression assays during development of fibrosis. The results are an incremental advance of interest to a limited number of investigators. The methods appear technically sound and the data might prove to have some utilitarian value. ****** 6. 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 ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0276215.r001
Revision 1
29 Sep 2022 Author Response We highly appreciate the opportunity to improve our manuscript according to the valuable comments highlighted by the Reviewers. We have done our best to fully respond to the concerns raised by the reviewers. Reviewer #1: This paper presents proper reference genes for the RT-qPCR in the pulmonary fibrosis mouse model after treatment of the Bleomycin. In the fibrosis mouse model, the reference gene selection is an important and appropriate study for verifying gene expression. If the authors solve the minor issues below, this paper will be published to the PLoS One. 1) Manuscript Overall: Gene Symbol should be used as an italic, and all capital letters. RESPONSE: In our manuscript we have followed the gene nomenclature used by the National Center for Biotechnology Information (NCBI), see e.g., the gene for mus musculus collagen type I α1 chain (https://www.ncbi.nlm.nih.gov/gene/12842). According to this, mouse genes are written in a way that they start with an uppercase letter and all other letters are given by lowercase letters. This style is used to differentiate rodent genes from human genes, which are written in capital letters. Official gene symbols for mouse genes are provided by the Mouse Genome Informatics at the Jackson laboratory (http://www.informatics.jax.org/mgihome/nomen/gene.shtml#gaas), as follows: “Guidelines for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations in Mouse and Rat 2.3 Gene Symbols and Names 2.3.1 Gene Symbols Genes are given short symbols as convenient abbreviations for speaking and writing about the genes. A gene symbol should: • be unique within the species and should not match a symbol in another species that is not a homolog. • be short, normally 3-5 characters, and not more than 10 characters • use only Roman letters and Arabic numbers • begin with an uppercase letter (not a number), followed by all lowercase letters / numbers (see exception below) • not include tissue specificity or molecular weight designations • include punctuation only in specific special cases (see below) • ideally have the same initial letter as the initial letter of its gene name to aid in indexing. However, letter order in a gene symbol need not follow word order in the name. • When a definitive human ortholog exists, gene symbols should agree with human gene symbols when practical. • Never prepend 'm' (for a mouse gene symbol) or 'r' (for a rat gene symbol) to indicate species.” Based on this convention, we would like to stay with the original gene symbol nomenclature, if acceptable by the reviewer. All gene names were originally in italic, but we noticed that when submitting, the system converted italic font to not italic in the Abstract and we were not able to correct this. All gene names are (and were originally) in italic in the Word version of the manuscript. 2) Line 2: Please provide abbreviation of reverse transcription quantitative real-time PCR as (RT-qPCR) in title. RESPONSE: The abbreviation (RT-qPCR) has been added in the title; page 1, line 5. 3) Line 17: two separate algorithms → Please present specific names. RESPONSE: Names of two separate algorithms are now present in the Abstract, page 2, line 31. 4) Line 85-86: “ab libitum” please provide as italic. RESPONSE: “ab libitum” is provided as italic, page 5, lines 101-102. 5) Line 130 Table 1, 5th and 7th columns: please provide direction of primer sequences (5’-3’). RESPONSE: Directions of primer sequences (5’-3’) has been provided on 5th and 7th columns, page 7, Table 1, row 1. 6) Figure 3 and 5: There may be confusion in sample names. “Untreated” → “Untreated samples”, “Bleomycin” → “Bleomycin-treated samples”. If you lack space, you can use abbreviations. "UC" and "BTS". The difference between the groups according to the shape of each point (round point, square point) should be described in each figure legend. RESPONSE: The requested change “Untreated” → “Untreated samples”, “Bleomycin” → “Bleomycin-treated samples” has been made in the Fig 2 and Fig 5. Fig 3 itself has not been changed but the following explanation; “Untreated: untreated samples. Bleomycin: bleomycin-treated samples” has been added in the Fig 3 legend, page 10, line 219. The difference between the groups according to the shape of each point; untreated (round symbols) and bleomycin-treated (square symbols) is now described in the Fig 1 (page 9, lines 188-189), 2 (page 9, lines 199-200) and 5 (page 11, lines 242-243) legends. Reviewer #2: The ms. by Norman et al. presents data regarding a number of genes that could serve as reference genes in the bleomycin mouse model. These are proposed as stable denominators for gene expression assays during development of fibrosis. The results are an incremental advance of interest to a limited number of investigators. The methods appear technically sound and the data might prove to have some utilitarian value. https://doi.org/10.1371/journal.pone.0276215.r002
2 Oct 2022 Decision Letter - Kyung-Wan Baek, Editor Identification of suitable reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) in the fibrotic phase of the bleomycin mouse model of pulmonary fibrosis PONE-D-22-22612R1 Dear Dr. Heikkinen, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Kyung-Wan Baek, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): It appears that the manuscript has been improved to a level suitable for publication in PLoS ONE. I do not believe that further review is necessary. Reviewers' comments: https://doi.org/10.1371/journal.pone.0276215.r003
Formally Accepted
7 Oct 2022 Acceptance Letter - Kyung-Wan Baek, Editor PONE-D-22-22612R1 Identification of suitable reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) in the fibrotic phase of the bleomycin mouse model of pulmonary fibrosis Dear Dr. Heikkinen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Kyung-Wan Baek Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0276215.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .