Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae

Saqib Rahman; Zhenzhen Zhao; Muhammad Umair Sial; Yanning Zhang; Hongyun Jiang

doi:10.1371/journal.pone.0258201

Peer Review History

Original SubmissionJanuary 17, 2021
18 Mar 2021 Decision Letter - Yonggen Lou, Editor PONE-D-21-00308 Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae PLOS ONE Dear Dr. Jiang, 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. ============================== I agree with the comments proposed by the two reviewers. Please improve the manuscript according to their suggestions. ============================== Please submit your revised manuscript by Apr 25 2021 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. 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Reviewer #1: Yes Reviewer #2: Partly ******** 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: No ****** 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 manuscript written by Rahman et al. reported that actin but not 18s can be used to analyze gene expression at development stages of Myzus persicae. This is a basic work but is very important for studying molecular biology of M. persicae. Overall, the experiments were well designed and the manuscript was well written. I only have a few minor concerns: 1) In page 4, line 3, five methods should be revised as one method and three software. 2) In figure 2, 3 and 4, please put “Actin”, “18S”, “Actin and 18S” into each panel. This will make the figures clear. 3) In page 9, line 18, you should give the full name when first used. 4) In page 17, line 24, “under the same treatment of one insecticide”? Reviewer #2: This manuscript by Rahman et al. validated the stability of two reference genes Actin and 18S by analyzed the relative expression levels of four target genes. I have some questions for this research. First, it is better to standardize the nomenclature associated with quantitative PCR to avoid confusion; for example, the abbreviation RT-PCR should be used for reverse transcription PCR, qPCR should be used for quantitative real-time PCR, and RT-qPCR should be used for reverse transcription-qPCR. However, in this study, the abbreviations relative to PCR is very confusing; there are four types of abbreviation, "real-time qRT-PCR", "real-time RT-qPCR", "RT-qPCR" and "qRT-PCR". Second, since the purpose of this study is to validate the stability of two reference genes, it is necessary to show all the Ct values if possible. Third, consider to rearrange Figure 2-4. It is hard for reader to compare the tendency of the relative expression levels of one target gene (normalized to Actin, 18S, or two-gene combination). I think it is better to put the three results of one same gene (normalized to Actin, 18S, or two-gene combination) into an independent line chart. Then combine the four graphs (four target genes that with the same treatment) together. Fourth, it is not nice for reviewer to make comment when the line number of each page is independent. In addition, the followings should be considered in revising the manuscript. Page 2, line 13: The initial of actin should be capitalized. Please check the full manuscript. Page 3, line 9: quantify gene expression Page 4, Line1: the appropriate abbreviation of ribosomal protein L27 should be RPL27. And RPL32 should be italic. Line 10-12: why you chose this four target genes has to be mentioned in introduction. Line 21: need format modification. Please check the full manuscript. Page 5, Line 11: Adult aphids used in the study are female only, male only, or random. And how many days have the adults emerged. Line 24, 25: The cDNAs have been synthesized by reverse transcription PCR, hence, only qPCR here. It has to be clarified if the "qRT-PCR" here stands for "quantitative real-time PCR". Page 6, Line 9: Pfaffl [6] Line 26-28: It is better to count the numbers to same decimal places. Page 7, Line 3: The mortalities were too low and have no differences among three concentrations. It might be exposure to insecticides only for 24 h or the concentrations of these two insecticides might be not appropriate. Besides, the mortality data of control was not listed in Table 2. Line 10, 26: I noticed that the differences only occurred at one or two tested time point, and not all target genes show differences when normalized with Actin, 18S, or two-gene combination. It seems that the difference occur relative to the expression profile of different target gene or the deviation among samples. These results were not persuasive to evaluate the stability of the reference gene. Page 8, Line 8: Define Ct. Besides, for example, 16.51±1.51 indicated mean ± standard error or standard deviation. Page 9, Line 18: Define RPS, TATA, HSP and SDHA. Page 10, Line 12: The reference genes were ranked from the most stable to the least stable, but the "<" here was very confusing when lack of enough background. I think it is not appropriate to copy exactly the description in the reference. ****** 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. 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Revision 1
2 May 2021 Author Response Response to Reviewer 1 Reviewer #1: The manuscript written by Rahman et al. reported that actin but not 18s can be used to analyze gene expression at development stages of Myzus persicae. This is a basic work but is very important for studying molecular biology of M. persicae. Overall, the experiments were well designed and the manuscript was well written. I only have a few minor concerns: Response: We are very grateful to your comments on the manuscript. At the same time, we have thoroughly revised the manuscript in order to express the idea more clearly. We believe the manuscript has been greatly improved. Once again, thank you for the kind advice and acknowledgments. 1. In page 4, line 3, five methods should be revised as one method and three software. Response: DONE. “…five methods…” was changed to “one method and three software”. 2. In figure 2, 3 and 4, please put “Actin”, “18S”, “Actin and 18S” into each panel. This will make the figures clear. Response: DONE. “Actin”, “18S” and “Actin and 18S” were added into each panel. 3. In page 9, line 18, you should give the full name when first used. Response: DONE. The full name was added for each gene when it was used firstly, as follow. “…..ribosomal protein L (RPL), Tublin, GADPH, ribosomal protein S (RPS), 18S, EF1A, TATA box binding protein (TATA), heat shock protein (HSP) and succinate dehydrogenase complex subunit A (SDHA)”. 4. In page 17, line 24, “under the same treatment of one insecticide”? Response: DONE. Page 14, lines 15-18, “among different concentrations…under the same treatment of one insecticide…” was changed to “…..among different starvation time…..under the same starvation time”. Page 14, lines 23-26, “among different concentrations…under the same treatment of one insecticide…” was changed to “among different developmental stages ….under the same developmental stage”. Response to Reviewer 2 Reviewer #2: This manuscript by Rahman et al. validated the stability of two reference genes Actin and 18S by analyzed the relative expression levels of four target genes. I have some questions for this research. Response: We are very grateful to your comments on the manuscript. At the same time, we have thoroughly revised the manuscript in order to express the idea more clearly. We believe the manuscript has been greatly improved. Once again, thank you for the kind advice and acknowledgments. 1. First, it is better to standardize the nomenclature associated with quantitative PCR to avoid confusion; for example, the abbreviation RT-PCR should be used for reverse transcription PCR, qPCR should be used for quantitative real-time PCR, and RT-qPCR should be used for reverse transcription-qPCR. However, in this study, the abbreviations relative to PCR is very confusing; there are four types of abbreviation, "real-time qRT-PCR", "real-time RT-qPCR", "RT-qPCR" and "qRT-PCR". Response: DONE. We checked this manuscript carefully and standardized the nomenclature associated with quantitative PCR to only one type, RT-qPCR. 2. Second, since the purpose of this study is to validate the stability of two reference genes, it is necessary to show all the Ct values if possible. Response: DONE. The mean Ct and standard error values of of Actin and 18S for all samples in this paper were shown in the section of “Expression of target genes normalized to Actin and 18S at different developmental stages of M. persicae”. 3. Third, consider to rearrange Figure 2-4. It is hard for reader to compare the tendency of the relative expression levels of one target gene (normalized to Actin, 18S, or two-gene combination). I think it is better to put the three results of one same gene (normalized to Actin, 18S, or two-gene combination) into an independent line chart. Then combine the four graphs (four target genes that with the same treatment) together. Response: DONE. Thanks for your good suggestion. We reviewed Figures 2-4 according to the suggestions of Reviewer 1. We added “Actin”, “18S”, “Actin and 18S” into each panel. 4. Fourth, it is not nice for reviewer to make comment when the line number of each page is independent. Response: DONE. We have numbered each line of this paper continuously. In addition, the followings should be considered in revising the manuscript. 5. Page 2, Line 13: The initial of actin should be capitalized. Please check the full manuscript. Response: DONE. We have checked carefully the full manuscript and corrected. 6. Page 3, line 9: quantify gene expression. Response: DONE. “….quantity RNA expression…” was changed to “….quantity gene expression…”. 7. Page 4, Line1: the appropriate abbreviation of ribosomal protein L27 should be RPL27. And RPL32 should be italic. Response: DONE. “L27” was changed to “RPL27” and RPL32 was in italic type. Line 10-12: why you chose this four target genes has to be mentioned in introduction. Response: DONE. We have given the reasons as follow: CYP6CY3 and E4 are belong to metabolic enzymes, which have been studied widely and deeply and associated to insecticide resistance in M. persicae [1]. The over-expression of these two genes induced by insecticide treatment even as sublethal doses have been observed in many insects including M. persicae [20-22]. CYP6-1 is a member of cytochrome 450 monooxygenase family. VPS11 is involved in vesicle transport to vacuoles which plays an important role in segregation of intracellular molecules into distinct organelles [23]. However, both CYP6-1 and VPS11 are unfamiliar in M. persicae. The changes in the expression pattern of these two genes induced by the changes of both biotic and abiotic conditions would be unpredictable, which would give a relative comparison. Line 21: need format modification. Please check the full manuscript. Response: DONE. “….a 16 L:8D photoperiod…” was changed to “….a 16 Light: 8 Dark photoperiod…”. And We check the full manuscript carefully. 8. Page 5, Line 11: Adult aphids used in the study are female only, male only, or random. And how many days have the adults emerged. Response: DONE. This section was change to “Five different growing stages of M. persicae (green peach aphids), including 1st, 2nd, 3rd, and 4th instar nymphs and newly emerged adults, were collected as samples [20]. For each nymph sample, a total of 60 aphids were collected, and for each adult sample, total random 30 aphids were collected.” Line 24, 25: The cDNAs have been synthesized by reverse transcription PCR, hence, only qPCR here. It has to be clarified if the "qRT-PCR" here stands for "quantitative real-time PCR". Response: DONE. We checked this manuscript carefully and standardized the nomenclature associated with reverse transcription PCR and quantitative real-time PCR. 9. Page 6, Line 9: Pfaffl [6] Response: DONE. “Pfaffl6” was changed to “Pfaffl [6]”. Line 26-28: It is better to count the numbers to same decimal places. Response: DONE. The data was unified to the same decimal places. 10. Page 7, Line 3: The mortalities were too low and have no differences among three concentrations. It might be exposure to insecticides only for 24 h or the concentrations of these two insecticides might be not appropriate. Besides, the mortality data of control was not listed in Table 2. Response: DONE. Firstly, the mortality data of control group was listed in Table 2. Secondly, the population used in this study is a field population collected from Nanping, Fujiang Province in 2018 (Sial et al., 2020, [3]), which have developed a certain level of resistance to pesticides. Therefore, the concentrations of tested two insecticides used this study just induced sublethal effects. And the highly mortalities were not the aim of this study. The metabolic resistance mechanism related to the over-production of carboxylesterases E4 and Cytochrome P450 monooxygenases CYP6CY3 seem to confer low to moderate levels of resistance to pesticides in Myzus persicae, which have been documented in a lot of paper. In a word, the changes in the expression of metabolic enzymes induced by sublethal-doses of pesticides were detected in this paper. Line 10, 26: I noticed that the differences only occurred at one or two tested time point, and not all target genes show differences when normalized with Actin, 18S, or two-gene combination. It seems that the difference occur relative to the expression profile of different target gene or the deviation among samples. These results were not persuasive to evaluate the stability of the reference gene. Response: DONE. This section was changed to “As shown in Fig 3, the tendency of expression changes of the four target genes normalized to Actin was generally consistent with those normalized to 18S and was initially inhibited and then promoted with changes in starvation time (Figs 3A and B). After starvation for 6h, the relative expression levels of four target genes were all inhibited significantly compared to those without starvation treatment (starvation for 0 hours) normalized both with Actin and 18S as reference gene respectively, except for E4 and VSP11 normalized with Actin. The relative expression levels of four target genes were all increased significantly in M. persicae after starvation for 48h normalized both with Actin and 18S as reference gene respectively. A comparison between the relative expression levels normalized with Actin and 18S showed that significant differences occurred for all tested target genes, with CYP6CY3 at 12 h and 48 h, CYP6-1 at 6 h, E4 at 12 h, and VPS11 at 6 h and 12 h. As shown in Fig 3C, the expression fold normalized to the combination of Actin and 18S showed no differences compared with those normalized to 18S individually (Figs 3B and C).” 11. Page 8, Line 8: Define Ct. Besides, for example, 16.51±1.51 indicated mean ± standard error or standard deviation. Response: DONE. The define “threshold cycle” was added for Ct. And “….the Ct values….” were changed to “mean threshold cycle (Ct) and standard error values” in lines 8 and 10. 12. Page 9, Line 18: Define RPS, TATA, HSP and SDHA. Response: DONE. The full name for RPS, TATA, HSP and SDHA were added. 13. Page 10, Line 12: The reference genes were ranked from the most stable to the least stable, but the "<" here was very confusing when lack of enough background. I think it is not appropriate to copy exactly the description in the reference. Response: DONE. It was changed to “>”. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0258201.r002
25 Jun 2021 Decision Letter - Yonggen Lou, Editor PONE-D-21-00308R1 Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae PLOS ONE Dear Dr. Jiang, 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 improve the manuscript according to the suggestion of the reviewer #2. ============================== Please submit your revised manuscript by Aug 09 2021 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. 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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. [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: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 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: (No Response) Reviewer #2: The followings should be still considered in revising the manuscript. 1. Line 169 and 173, add a space between number and "h". 2. Line 206, italicize the "E". 3. Line 207, there is no Fig 5 in this manuscript. 4. Line 277, delete the space between "-" and "q". ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [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.0258201.r003
Revision 2
28 Jul 2021 Author Response Response to Reviewer 2 Reviewer #2: The followings should be still considered in revising the manuscript. Response: We are very grateful to your comments on the manuscript. At the same time, we have thoroughly revised the manuscript in order to express the idea more clearly. We believe the manuscript has been greatly improved. Once again, thank you for the kind advice and acknowledgments. 1. Line 169 and 173, add a space between number and "h". Response: DONE. A space was added between number and “h”. 2. Line 206, italicize the "E". Response: DONE. The “E”was changed to “E”. 3. Line 207, there is no Fig 5 in this manuscript. Response: DONE. The Fig 5 was submitted. 4. Line 277, delete the space between "-" and "q". DONE. The space was deleted between "-" and "q". Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0258201.r004
22 Sep 2021 Decision Letter - Yonggen Lou, Editor Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae PONE-D-21-00308R2 Dear Dr. Jiang, 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, Yonggen Lou Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0258201.r005
Formally Accepted
8 Oct 2021 Acceptance Letter - Yonggen Lou, Editor PONE-D-21-00308R2 Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae Dear Dr. Jiang: 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. Yonggen Lou Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258201.r006

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