Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness

Amanda Formosa; Erica Acton; Amy Lee; Paul Turgeon; Shehla Izhar; Pamela Plant; Jim N. Tsoporis; Sabri Soussi; Uriel Trahtemberg; Andrew Baker; Claudia C. dos Santos

doi:10.1371/journal.pone.0286871

Peer Review History

Original SubmissionFebruary 8, 2023
16 Mar 2023 Decision Letter - Jacopo Sabbatinelli, Editor PONE-D-23-03677Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illnessPLOS ONE Dear Dr. Formosa, 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 Apr 30 2023 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. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 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 ****** 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 ****** 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: Formosa et al. explore potential candidate as endogenous controls for miRNA evaluation in the critically ill patients. The manuscript is of interest. Some comments: Abstract. I would highlight the potential of miRNAs as biomarkers with potential clinical application. One of the consequences of your results is the translation to the clinical practice could be facilitated with the miRNA candidates. Introduction. I would highlight the impact of severe COVID-19 in more vulnerable groups elderly patients, with high level of comorbidities, immunodeficiency, ... Please, include some data on the high rate of mortality among critically ill patients. In addition, I would not only focus on the immune response. The adverse outcomes are caused by different mechanisms, immune response, inflammation, poor repair, ... Due to its nature, miRNAs would provide information on all these mechanisms. I would also introduce the use of miRNAs as biomarkers for adverse outcomes. The comparison COVID-19 vs healthy controls is not clinically relevant. Please, provide additional data on the criteria used. For instance, what percentage of expression, related to the number of samples, did you establish? It should 100 % for both study phases. Did you establish a maximum Cq? Authors should compare the levels of miRNAs according to the characteristics of the study population. Are there differences between sex, presence of comorbidities (hypertension, diabetes, …), treatments (corticoids, ventilatory support, …)? Do miRNAs correlate with age? Discussion should be completed with different topics. Are these candidates biomarkers in COVID-19? Could these miRNAs be quantified in other type of samples such as serum or plasma? Could you evaluate the impact of the hemolysis in your samples? “RNA concentration of the samples was determined using ThermoFisher Qubit system with RNA 140 HS assay kit.” It is repeated. Which criteria did you use to evaluate duplicates? When did you repeat the qPCR? Due to the huge problem in reproducibility, please, provide detailed information on the RNA isolation and RT-qPCR protocols. At least as supplemental information. Please, use the appropriate statistical test to compare both cohorts. I would add a Limitations paragraph Please, add additional information to the Figure Legend. ****** 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 ******** [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.0286871.r001
Revision 1
12 May 2023 Author Response May 9th 2023 Dear PLOS ONE editorial board, Please find below our detail point by point response to the editor and reviewer’s comment for our manuscript entitled, “Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness”. The editor/reviewer’s comments are listed and our response is directly below each comment. Please note that line numbers refer to the revised manuscript with track changes. 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 Response: we have accessed both urls and edited our manuscript in detail to reflect the formatting guidelines 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. Response: we obtained written consent from substitute decision makers and this is now updated in the methods section (lines 185-186) 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Response: we agree that data sharing provides the best way for readers to fully assess and reproduce our results. The data is now included in Supplementary Tables (S1 – S4 Table) and updated in the manuscript file (lines 724-730) 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Response: we now have our data compiled in Supplementary Tables S1-S4. 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: Response: the ORCID ID for the submitting and corresponding author (Dr. A. Formosa) has been validated. I (A. Formosa) have contacted the editorial office regarding adding a second corresponding author, Dr. dos Santos. I was advised a second corresponding author is permitted and this is now reflected in the manuscript. I did not find a way to insert Dr. dos Santos’ ORCID ID in the system but it is: https://orcid.org/0000-0002-6446-8791 Reviewer #1: Formosa et al. explore potential candidate as endogenous controls for miRNA evaluation in the critically ill patients. The manuscript is of interest. Some comments: Abstract. I would highlight the potential of miRNAs as biomarkers with potential clinical application. One of the consequences of your results is the translation to the clinical practice could be facilitated with the miRNA candidates. Response: Thank you for this comment and the abstract has been adjusted accordingly (lines 71-72) Introduction. I would highlight the impact of severe COVID-19 in more vulnerable groups elderly patients, with high level of comorbidities, immunodeficiency, ... Please, include some data on the high rate of mortality among critically ill patients. Response: This information has now been included with a relevant citation (lines 109-111) In addition, I would not only focus on the immune response. The adverse outcomes are caused by different mechanisms, immune response, inflammation, poor repair, ... Due to its nature, miRNAs would provide information on all these mechanisms. I would also introduce the use of miRNAs as biomarkers for adverse outcomes. The comparison COVID-19 vs healthy controls is not clinically relevant. Response: Thank you for this comment and we agree. Our introduction has been edited accordingly and we have deleted the comment on comparison to healthy controls, which we also believe to be clinically irrelevant (lines 128-130; 135-140) Please, provide additional data on the criteria used. For instance, what percentage of expression, related to the number of samples, did you establish? It should 100 % for both study phases. Did you establish a maximum Cq? Response: We agree that expression in all samples is of utmost importance given we are studying reference genes. We have alluded to that in the methods when discussing the screening of candidate genes for RT-qPCR. To highlight this importance further, we have edited lines 236-238. For candidate miRNAs included in reference gene selection algorithms, only those with 100% expression across all patient samples were included. We have now added more information on this, including minimum and maximum Cq values, in our results section (lines 401-403). Authors should compare the levels of miRNAs according to the characteristics of the study population. Are there differences between sex, presence of comorbidities (hypertension, diabetes, …), treatments (corticoids, ventilatory support, …)? Do miRNAs correlate with age? Response: The Ct values of the 4 candidate reference miRNAs (each averaged over two technical replicates) were tested for associations with the following study population characteristics in the validation cohort: sex, age, the presence of comorbidities (hypertension, type II diabetes, dyslipidemia) and treatments (antibiotics, steroid use, respiratory support). For binary metadata (2 groups), miRNA expression was compared using a Welch's 2-sided t-test. Categorical metadata with 3 or more groups was modelled against miRNA expression using ANOVA. Continuous parameters were modelled using linear regression. No significant associations (p < 0.05) were found between any reference miRNA and the study population characteristics tested. The model outputs and p-values are now included in Supplemental Table S1. The description of these tests are now included in the Methods at lines 282-294, and referenced in the Discussion at lines 525-527. Discussion should be completed with different topics. Are these candidates biomarkers in COVID-19? Response: we reviewed the literature and did not find any evidence of these miRNAs as biomarkers in COVID-19 critical illness. This is now outlined in the discussion lines 495-500. Could these miRNAs be quantified in other type of samples such as serum or plasma? Response: we have commented on the potential of these miRNAs to be tested as candidate reference genes in different sample types in our new limitations paragraph (lines 529-539) Could you evaluate the impact of the hemolysis in your samples? Response: our new limitations paragraph addresses this concern in not knowing the origin of the miRNA signals (inherent issue to using Paxgene tubes, lines 530-532) “RNA concentration of the samples was determined using ThermoFisher Qubit system with RNA 140 HS assay kit.” It is repeated. Response: thank you for pointing this out. The redundant phrase has been deleted (lines 205-211). Which criteria did you use to evaluate duplicates? When did you repeat the qPCR? Response: duplicates were average only if Ct data were within 0.5 cycles of each other. All technical replicates for all samples for a given gene (sample maximization method) were run on the same qPCR plate. This is now noted in the materials and methods sections lines 267-268 and 280-281. Due to the huge problem in reproducibility, please, provide detailed information on the RNA isolation and RT-qPCR protocols. At least as supplemental information. Response: we have peeled through our materials and methods section and added more detailed information to ensure reproducibility (lines 184-281) Please, use the appropriate statistical test to compare both cohorts. Response: we have included further statistical testing as requested above including the analysis of the four candidate reference miRNAs across population study characteristics. Our input data for GeNorm, Normfinder, Bestkeeper and RefFinder was Ct values across all patients as required by the algorithms (S3 – S4 tables). I would add a Limitations paragraph Response: this has been added (lines 529-539) Please, add additional information to the Figure Legend. Response: further information regarding the rankings and Y axis values has been added (lines 461-464) Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0286871.r002
25 May 2023 Decision Letter - Jacopo Sabbatinelli, Editor Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness PONE-D-23-03677R1 Dear Dr. Formosa, 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, Jacopo Sabbatinelli, MD, PhD 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: (No Response) ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: The authors have properly addressed all comments. The manuscript has been substantially improved and deserves publication. ****** 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 ******** https://doi.org/10.1371/journal.pone.0286871.r003
Formally Accepted
21 Aug 2023 Acceptance Letter - Jacopo Sabbatinelli, Editor PONE-D-23-03677R1 Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness Dear Dr. Formosa: 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. Jacopo Sabbatinelli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0286871.r004

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