MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health

Lene B. Dypås; Kristine B. Gützkow; Ann-Karin Olsen; Nur Duale

doi:10.1371/journal.pone.0231040

Peer Review History

Original SubmissionDecember 19, 2019
6 Feb 2020 Decision Letter - Zhong-Cheng Luo, Editor PONE-D-19-35144 MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health PLOS ONE Dear Miss Dypås, 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 comments from the two reviewers carefully, and detail where/what changes have been made. We would appreciate receiving your revised manuscript by Mar 22 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. 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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 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Dypas et. al. investigated the quality of a specific cohort of bio-banked plasma samples and the ability to reproducibly isolate miRNA from these samples for downstream quantitative analysis. Utilizing three independent techniques for determining plasma quality, they demonstrate that their plasma samples are of high quality (low MP, low hemolysis) and can be used for quantification of circulating miRNA levels. These experiments were properly controlled, and the manuscript is nicely written. My only major comment is the following: Although the authors mention performing correlation analysis between each miRNA for the 38 pairs, these data are not included in manuscript. As the data in Figure 4 suggests that there may actually a large group of miRNAs with higher expression levels (>log2(0)) that correlate between cord and maternal samples, it would be important to include the R2 and p-values for each miRNA correlation as a supplement in the manuscript. P-value for the linear regression analysis in Figure 4 should also be included. Minor comments (1) In methods, the authors state that they collected 50 pairs of cord blood/maternal blood. However, there are only 38 pairs included in the miRNA analysis, with no explanation for the exclusion of other sample pairs. Please include a statement on why 12 pairs were excluded from miRNA analysis, as this could be an important technical caveat. (2) No statement of patient consent prior to sample collection. This should be included. (3) Figure 3: Individual points for each sample should be shown in the box plots. (4) How might altered levels of exosomes affect miRNA analysis? Is there a difference in exosomes between cord blood and maternal blood? Reviewer #2: The authors conducted thorough examination of the quality of the collected plasma samples by assessing microparticle counts and hemeolysis; and profiled many miRNAs to identified those expressed in cord and maternal blood plasma. The overall analysis procedure was strict and correct cautions were taken to ensure high quality of the obtained data. The quality of the manuscript is suitable to be published in PLOSOne. One question: Line 180 : NRQ = 2-ΔCq (where ΔCq = ΔCq (target) - Cq (global mean)). Please confirm ΔCq (target) is used but not Cq (target); also “global mean” is not defined. ****** 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. 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Revision 1
9 Mar 2020 Author Response Reviewer comments, author responses and manuscript changes Reviewer #1 Comment 1: Although the authors mention performing correlation analysis between each miRNA for the 38 pairs, these data are not included in manuscript. As the data in Figure 4 suggests that there may actually a large group of miRNAs with higher expression levels (>log2(0)) that correlate between cord and maternal samples, it would be important to include the R2 and p-values for each miRNA correlation as a supplement in the manuscript. P-value for the linear regression analysis in Figure 4 should also be included. Response: We agree that this is valuable information and Figure 4 has been revised to include the p-value for the linear regression analysis, and, a new table (S2 table) containing the R2 and p-values for each miRNA correlation has been included in supplementary information. Comment 2: In methods, the authors state that they collected 50 pairs of cord blood/maternal blood. However, there are only 38 pairs included in the miRNA analysis, with no explanation for the exclusion of other sample pairs. Please include a statement on why 12 pairs were excluded from miRNA analysis, as this could be an important technical caveat. Response: Thank you for pointing out that this important piece of information was missing. The manuscript has been revised to include a statement explaining that 12 pairs were excluded due to insufficient sample volume (line 111-112). Comment 3: No statement of patient consent prior to sample collection. This should be included. Response: A statement of patient consent prior to sample collection has been included in the methods section (line 117-118). Comment 4: Figure 3: Individual points for each sample should be shown in the box plots. Response: Figure 3 has been revised to include individual points for each sample as we agree that this enhances the readability of the plot and adds value. Comment 5: How might altered levels of exosomes affect miRNA analysis? Is there a difference in exosomes between cord blood and maternal blood? Response: The questions raised here is a very interesting topic, with no clear answers as of yet. The manuscript has been revised to include a paragraph on miRNAs in exosomes to clarify (line 224-229). Early research hypothesized that extracellular miRNAs were protected by encapsulation in exosomes, and that the majority of miRNAs found in plasma or serum stemmed from these vesicles (1). Later it was discovered that 90-99% of extracellular miRNAs were actually associated with proteins in the argonaute family, not exosomes (2, 3). Moreover, it has also been shown that exosomes might actually contain very small amounts of miRNA, contrary to what was hypothesized before (4), and therefore might not affect analysis of total circulating miRNAs to such an extent as previously believed. Either way, when analyzing circulating miRNAs there is no precedence to remove exosomes from plasma, and miRNAs in exosomes are regarded as part of the circulating (i.e. extracellular) miRNAs, and altered levels of exosomes containing miRNAs then reflect changes in the individual’s physical state causing altered levels of circulating miRNAs. However, it is important to always include details of plasma preparation steps in your protocol so that readers know if exosomes are included in the plasma (or other sample material) for reproducibility and comparative purposes. Regarding your question on a difference in exosomes between cord and maternal blood, not much is known. There is however one small study on this in serum, where they found slightly higher concentrations of exosomes in serum from maternal blood compared to cord blood (5). Reviewer #2 Comment 1: Line 180 : NRQ = 2-ΔCq (where ΔCq = ΔCq (target) - Cq (global mean)). Please confirm ΔCq (target) is used but not Cq (target); also “global mean” is not defined. Response: Thank you for pointing this out. ΔCq(target) is a typo, and should be Cq(target) as you correctly assumed. The typo has been corrected and a definition of “global mean” has been added (line 183-184). References 1. Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS One. 2008;3(11):e3694-e. 2. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A. 2011;108(12):5003-8. 3. Turchinovich A, Weiz L, Langheinz A, Burwinkel B. Characterization of extracellular circulating microRNA. Nucleic Acids Res. 2011;39(16):7223-33. 4. Chevillet JR, Kang Q, Ruf IK, Briggs HA, Vojtech LN, Hughes SM, et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc Natl Acad Sci U S A. 2014;111(41):14888-93. 5. Jia L, Zhou X, Huang X, Xu X, Jia Y, Wu Y, et al. Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration. FASEB J. 2018;32(8):4534-43. Attachments Attachment Submitted filename: response_to_reviewers.docx https://doi.org/10.1371/journal.pone.0231040.r002
16 Mar 2020 Decision Letter - Zhong-Cheng Luo, Editor MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health PONE-D-19-35144R1 Dear Dr. Dypås, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Zhong-Cheng Luo Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0231040.r003
Formally Accepted
20 Mar 2020 Acceptance Letter - Zhong-Cheng Luo, Editor PONE-D-19-35144R1 MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health Dear Dr. Dypås: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With 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.0231040.r004

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