Quality assessment of RNA in long-term storage: The All Our Families biorepository

Nikki L. Stephenson; Kylie K. Hornaday; Chelsea T. A. Doktorchik; Andrew W. Lyon; Suzanne C. Tough; Donna M. Slater

doi:10.1371/journal.pone.0242404

Peer Review History

Original SubmissionAugust 4, 2019
20 Sep 2019 Decision Letter - Rania Mohamed Labib, Editor PONE-D-19-21994 Quality assessment of RNA in long-term storage: The All Our Families biorepository PLOS ONE Dear Ms Stephenson, 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. We would appreciate receiving your revised manuscript by Nov 04 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Rania Mohamed Labib, PhD Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf [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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: 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 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: The authors Claim (in the title and conclusion) to have studied quantity and quality of RNA isolated from blood stored in PAXgene Blood RNA Tube for 7-10 years. However, they present data on RNA quantity and puritiy (Ratio 260/280 nm). Purity of RNA is no measure of quality, which should be depicted as integrity. A more or less degraded RNA - mark of pure quality - may sow perfect purity. RNA quality should therefore be assessed e.g. by BioAnalyser or - if not available - electrophoresis. To make it even better, expression levels of a set of genes or Array Analysis will give even better evidence of good oerall and mRNA quality. The authors demonstrate statistically significant differences in purity of RNA isolated in 2011 and 2018, with purity rising over time, which is an overvaluation of statistics. This difference is probably statistically significant, but it is not relevant. A pure nucleic acid is generally considered to have an 260/280 ratio of around 2, which is the case at both time points. The conclusions the authors draw from their results are correct: RNA quantity and purity do not change with storage time of blood samples under the given conditions. But before concluding that the remaining samples show unchanged good quality for further studies this should be demonstrated by methods mentioned above. Reviewer #2: Nikki Stephenson et. al, in their manuscript ‘Quality assessment of RNA in long-term storage: The All Our Families biorepository’, addressed a very general but important issue of sample storage in biorepository. In this manuscript the authors have analyzed RNA from whole blood samples from a cohort from 2011 and compared its quality with the RNA samples from the cohort of 2018. The authors conclude that RNA quality does not decrease over long term storage and the conditions used for the collection of samples, storage and extraction are robust. Although the manuscript hold merit with the high sample number from the biorepository, I have following specific concerns about the manuscript. Specific comments 1. The RNA concentration and purity are measured on simple nano-drop; which are not specific and usually give variable results. It is necessary to have RNA integrity number (RIN) by a bioanalyzer which. Further, correlation of RIN to RNA Quality Score (RQS) can further enhance the authenticity of the data. 2. The conclusions drawn from the statistical analysis are biased, supporting the fact that RNA quality and purity are either better or improving with time. Although the authors themselves have mentioned that it is clinically insignificant. ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0242404.r001
Revision 1
4 Nov 2019 Author Response 1. Editor’s comment: “Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.” Authors’ response: Thank you for the comments. To the best of the authors’ knowledge this submitted revised manuscript adheres to the PLOS ONE's style requirements. The style of in-text citation has been altered to include square brackets and are utilizing the Vancouver style for referencing. Also, the files have been renamed as suggested. 2. Reviewer #1 comment: “The authors Claim (in the title and conclusion) to have studied quantity and quality of RNA isolated from blood stored in PAXgene Blood RNA Tube for 7-10 years. However, they present data on RNA quantity and puritiy (Ratio 260/280 nm).” Authors’ response: Thank you for your comments. We have adjusted each of the methods (line 115), and discussion (lines 244-260) surrounding RNA concentration and RNA optical absorbency to reflect when we are specifically discussing each. Also, we have relabelled each of the figures and tables to note specifically when we are discussing RNA concentration (yield) and RNA optical absorbency (purity). 3. Reviewer #1 comment: “Purity of RNA is no measure of quality, which should be depicted as integrity. A more or less degraded RNA - mark of pure quality - may sow perfect purity. RNA quality should therefore be assessed e.g. by BioAnalyser or - if not available - electrophoresis. To make it even better, expression levels of a set of genes or Array Analysis will give even better evidence of good oerall and mRNA quality.” Authors’ response: We understand that a 260/280 ratio is a measure of purity, however our consideration of quality was to discuss both RNA concentration and purity together. To address your comment we have included an analysis on a subset of samples for which we have RNA integrity numbers. Though this is a smaller sample size (n=164) over a shorter time span (25 months), we hope that this inclusion (lines 48-51, 73, 102-104, 107-108, 111, Table 2, 193-205, Figure 6, and 251-267), along with the previous response feature analysis of RNA concentration and purity will provide sufficient evidence for our conclusions that neither the quality nor quantity of RNA decreased over storage time. 4. Reviewer #1 comment: “The authors demonstrate statistically significant differences in purity of RNA isolated in 2011 and 2018, with purity rising over time, which is an overvaluation of statistics. This difference is probably statistically significant, but it is not relevant. A pure nucleic acid is generally considered to have an 260/280 ratio of around 2, which is the case at both time points.” Authors’ response: We agree that the observed increase in both RNA quantity and quality, though statistically significant, are not clinically important. However, we do not feel that this is an overvaluation of statistics, rather than this one piece of statistical evidence provided by the regression model output is driven primarily by an increase in precision of the estimates, and when the data is assessed visually through figures 4-6 this increase in precision can be seen. We have mentioned both in the results section (lines 165-166, 186-187, 204-205), as well as in the discussion section (lines 217, 244-260) that while the analyses show a statistically significant positive association between storage time and both purity and concentration, this association is not clinically significant. However we can conclude that there was no decrease in purity, concentration, or RIN over time. 5. Reviewer #1 comment: “The conclusions the authors draw from their results are correct: RNA quantity and purity do not change with storage time of blood samples under the given conditions. But before concluding that the remaining samples show unchanged good quality for further studies this should be demonstrated by methods mentioned above.” Authors’ response: At the request of the reviewer we have included further analysis on a subset of samples analysing the association between storage time and RIN (lines 48-51, 73, 102-104, 107-108, 111, Table 2, 193-205, Figure 6, and 251-267). However, we are unable at this time to assess the RIN after the same amount of storage as we were assessing with purity and yield due to logistic constraints. 6. Reviewer #2 comment: “The RNA concentration and purity are measured on simple nano-drop; which are not specific and usually give variable results. It is necessary to have RNA integrity number (RIN) by a bioanalyzer which. Further, correlation of RIN to RNA Quality Score (RQS) can further enhance the authenticity of the data.” Authors’ response: Thank you for your comments. We have included further analysis on a subset of samples analysing the association between storage time and RIN. Though this is a smaller sample size (n=164) over a shorter time span (25 months), we hope that this inclusion (lines 48-51, 73, 102-104, 107-108, 111, Table 2, 193-205, Figure 6, and 251-267) along with the previous response feature analysis of RNA concentration and purity will provide sufficient evidence for our conclusions that the quality of RNA does not decrease over storage time. Unfortunately, the authors’ did not have access to the electropherogram trace for each of these samples, thus the height of the 18s and 28s RNA peaks, the area under the 18s and 28s RNA peaks, and the fast region area are unknown. Without these values one cannot compute the RNA quality score (Copois et al, 2007 https://doi.org/10.1016/j.jbiotec.2006.07.032). As the reviewer noted in the comment, RIN and RQS have been correlated, and the algorithm proposed by Schroeder et al, 2006 (doi:10.1186/1471-2199-7-3) has long been used as a gold standard of RNA quality, therefore although the authors’ recognize the value of including the RQS we feel that the inclusion of the RIN does enhance the authenticity of the data even without the RQS. 7. Reviewer #2 comment: ‘The conclusions drawn from the statistical analysis are biased, supporting the fact that RNA quality and purity are either better or improving with time. Although the authors themselves have mentioned that it is clinically insignificant.’ Authors’ response: While the authors’ agree that the systematic error leading to increased precision over time would bias the resultant estimates produced via both the response feature analysis and the linear regression, we feel that our conclusions- that the purity, yield, and RIN, do not decrease as a function of storage time- are unbiased as we clearly state in our description of the results that the resultant positive association is not clinically significant (lines 165-166, 186-187, 204-205), and in our discussion (lines 217, 244-260) we further emphasize this point noting that it is attributable to increased precision as seen through visual assessment of the models in figures 4 through 6, and potentially residual confounding. To accentuate this we have included an additional paragraph within the discussion (lines 244-260) outlining the influence of precision on the analysis and we offer our interpretation of our analysis. https://doi.org/10.1371/journal.pone.0242404.r002
11 Mar 2020 Decision Letter - Zhong-Cheng Luo, Editor PONE-D-19-21994R1 Quality assessment of RNA in long-term storage: The All Our Families biorepository PLOS ONE Dear Ms Stephenson, 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 address the additional comments from reviewer 1. We would appreciate receiving your revised manuscript by Apr 25 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Zhong-Cheng Luo Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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: I Don't Know ****** 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 now corrected RNA quality for puritiy and quantity, and give plausible explanations for the apparent increase in purity over time. These changes are statistically significant, but not clinically relevant. "Clinically significant" should be changed for "clinically relevant", or even better for "relevant", since this is rather a technical than a clinical aspect. Passages discussion the statistically evident seeming improvements should be shortened. Furthermore, to address RNA qualitiy, the authors included RIN numbers for a subset of samples (please change "subsample" for "subset of samples" , line 244), demonstrating no drop in overall RNA quality. Unfortunately, the storage time of the samples tested for RIN ranges only over 24 month as compared to 94 months for the purity and quantity measures. It would be nice to see RIN results at least for a small subset of samples after medium and long term storage (eg. 50 and 90 months. ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0242404.r003
Revision 2
8 Sep 2020 Author Response Reviewers' comments: The authors now corrected RNA quality for puritiy and quantity, and give plausible explanations for the apparent increase in purity over time. These changes are statistically significant, but not clinically relevant. "Clinically significant" should be changed for "clinically relevant", or even better for "relevant", since this is rather a technical than a clinical aspect. Passages discussion the statistically evident seeming improvements should be shortened. Furthermore, to address RNA qualitiy, the authors included RIN numbers for a subset of samples (please change "subsample" for "subset of samples" , line 244), demonstrating no drop in overall RNA quality. Unfortunately, the storage time of the samples tested for RIN ranges only over 24 month as compared to 94 months for the purity and quantity measures. It would be nice to see RIN results at least for a small subset of samples after medium and long term storage (eg. 50 and 90 months. Authors’ response: The Authors’ thank Reviewer 1 for the additional comments. As per request we have altered our terminology “clinically significant” to “technically relevant”, and we have decreased the discussion concerning statistical significance. Please see lines 50, 182, 204, 228, 240, and 281. To address the second part of the comment, we have included an additional 45 RINs with a long-term storage of 111-130 months. We are unable to complete an analysis at 50-90 months, as all samples had been stored past that time. We modeled the short-term storage (11-25 month) RINs and the long-term storage (111-130 months) RINs separately to ensure we would not introduce heterogeneity into a combined model. The most parsimonious model including all RIN data is reported, with the same conclusion as previously reported. We hope that this addition increases the reviewers confidence in the results regarding sample integrity. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0242404.r004
22 Sep 2020 Decision Letter - Zhong-Cheng Luo, Editor PONE-D-19-21994R2 Quality assessment of RNA in long-term storage: The All Our Families biorepository PLOS ONE Dear Dr. Stephenson, 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 Nov 06 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Zhong-Cheng Luo Academic Editor PLOS ONE Additional Editor Comments (if provided): There are 2 tracking-changes version in the same downlowned R2 PDF file, confusing ! Line 103, delete "a" before"another'. Table 2: please present all values to the precision of 2 decimal points. 3 decimal points are unnecessary. Line 50, Line 166 and Line 208, delete "technically". The word "relevant" is sufficient, and please do use the expression "technicically relevant" throughout the paper. Line 167, delete the confusing "(greater than for purity" [Note: HTML markup is below. Please do not edit.] [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.0242404.r005
Revision 3
26 Oct 2020 Author Response Comment: There are 2 tracking-changes version in the same downlowned R2 PDF file, confusing ! Response: This has been corrected. Comment: Line 103, delete "a" before"another'. Response: The text has been removed. Comment: Table 2: please present all values to the precision of 2 decimal points. 3 decimal points are unnecessary. Response: The table values have been changed to accommodate this request. Comment: Line 50, Line 166 and Line 208, delete "technically". The word "relevant" is sufficient, and please do use the expression "technicically relevant" throughout the paper. Response: The term has been changed throughout the document. Comment: Line 167, delete the confusing "(greater than for purity" Response: The text has been removed. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0242404.r006
29 Oct 2020 Decision Letter - Zhong-Cheng Luo, Editor PONE-D-19-21994R3 Quality assessment of RNA in long-term storage: The All Our Families biorepository PLOS ONE Dear Dr. Stephenson, 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 Dec 13 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Zhong-Cheng Luo Academic Editor PLOS ONE Additional Editor Comments (if provided): Page 9, line 185, the donfidence interval does not make sense : "-0.090 μg /tube (95% CI:-1.14, -0.67)", please check and correct the numbers. Edits: Page 9, line 194, replace "deline" with "decline" Page 10, line 206, replace "0.03" with "0.003" [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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.0242404.r007
Revision 4
29 Oct 2020 Author Response Editors comment: Page 9, line 185, the donfidence interval does not make sense : "-0.090 μg /tube (95% CI:-1.14, -0.67)", please check and correct the numbers. Authors’ Response: This has been corrected to -0.90 μg /tube (95% CI:-1.14, -0.67) Editors comment: Page 9, line 194, replace "deline" with "decline" Authors’ Response: This has been corrected. Editors comment: Page 10, line 206, replace "0.03" with "0.003" Authors’ Response: This has been corrected. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0242404.r008
3 Nov 2020 Decision Letter - Zhong-Cheng Luo, Editor Quality assessment of RNA in long-term storage: The All Our Families biorepository PONE-D-19-21994R4 Dear Dr. Stephenson, 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, Zhong-Cheng Luo Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0242404.r009
Formally Accepted
20 Nov 2020 Acceptance Letter - Zhong-Cheng Luo, Editor PONE-D-19-21994R4 Quality assessment of RNA in long-term storage: The All Our Families biorepository Dear Dr. Stephenson: 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. Zhong-Cheng Luo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0242404.r010

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