Understanding the qPCR Standard Curve: From Assay Validation to Absolute Quantification and Variance PCR
, Stephen A. Bustin 5, Jose M. Andrade 6, Robert Sjöback 7, Björn Sjögreen 3 and Anders Ståhlberg 2,8,9,10
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsMikael Kubista and the co-authors submitted a tutorial to the Int. J. Mol. Sci., devoted to the construction, validation, and analysis of qPCR standard curves.
The reviewer suggests some issues should be resolved before the manuscript might be recommended for publication.
1) The title is too narrow; it should be expanded to be more descriptive. Perhaps at lease "Tutorial" should be added?
2) The term "PCR" should be deciphered in the Abstract.
3) Please, add the name of the instrument and software used to obtain the amplification curves and standard curve to the description of Figure 1.
4) Figure 2 is truncated and should be moved to the next page.
5) There is no in-text reference to Figure 9.
6) Expand RSD in the caption to Table 1.
7) Subsection 3.1 duplicates the title of Section 3. Please, change the title of Subsection 3.1
8) Please, check lines 372, 375, 432, 462: Error! Bookmark not defined.
9) In-text references to Figures 7, 8, 12, 13, 14, 15, 16, 17, and 18 are placed after the figures.
10) Table 2 is inserted as a figure.
11) In the Conclusions section, add 1–2 sentences about how the results of this work can be used in clinical diagnostics or standards development.
12) In the Conclusions section, the authors refer to their proposed vPCR model, the corresponding section is 3.7. The authors do not mention the significance of this approach in either the Abstract or the Introduction. In this case, vPCR should probably be highlighted in a separate section rather than a subsection.
Author Response
1) The title is too narrow; it should be expanded to be more descriptive. Perhaps at lease "Tutorial" should be added?
Changed to: Understanding the qPCR standard curve: from assay validation to absolute quantification and variance PCR.
2) The term "PCR" should be deciphered in the Abstract.
Done
3) Please, add the name of the instrument and software used to obtain the amplification curves and standard curve to the description of Figure 1.
Amended
4) Figure 2 is truncated and should be moved to the next page.
Amended
5) There is no in-text reference to Figure 9.
Amended
6) Expand RSD in the caption to Table 1.
Amended
7) Subsection 3.1 duplicates the title of Section 3. Please, change the title of Subsection 3.1
Amended
8) Please, check lines 372, 375, 432, 462: Error! Bookmark not defined.
Amended
9) In-text references to Figures 7, 8, 12, 13, 14, 15, 16, 17, and 18 are placed after the figures.
MDPI style now applied
10) Table 2 is inserted as a figure.
Amended
11) In the Conclusions section, add 1–2 sentences about how the results of this work can be used in clinical diagnostics or standards development.
Paragraph 3 added.
12) In the Conclusions section, the authors refer to their proposed vPCR model, the corresponding section is 3.7. The authors do not mention the significance of this approach in either the Abstract or the Introduction. In this case, vPCR should probably be highlighted in a separate section rather than a subsection.
Good idea; done!
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript presents a detailed tutorial on how to build, validate, and analyze qPCR standard curves. By using an idealized standard curve with many replicates, the authors clearly explain basic concepts like PCR efficiency, LOD, and LOQ. They also introduced a theoretical idea called "variance PCR" (vPCR) to estimate target amounts using the variation in Cq. This article is highly significant because it corrects common mistakes in routine qPCR analysis and provides a strong educational guide for researchers to improve their assay quality and data reliability. Therefore, the manuscript is recommended to be accepted by the International Journal of Molecular Sciences. Here are some concerns for the authors to improve the quality of the manuscript:
Major concerns:
1) The authors introduce the concept of vPCR to estimate target copy numbers. While this is an interesting theoretical idea, its practical use is not very clear. Please add more discussion on the limitations of vPCR in real-world settings. Can a normal laboratory use this method easily? What specific equipment or conditions are required to make vPCR work reliably?
2) The "extreme" standard curve in the paper is built using purified synthetic DNA. However, real clinical or environmental samples often contain inhibitors that affect PCR performance. Please add a section or some examples discussing how complex sample matrices (like blood or tissue extracts) impact the LOD, LOQ, and the overall shape of the standard curve.
3) Even though I applaud the authors for vPCR. However, the authors seem like not influent in English writing. Therefore, I recommend the authors polish the whole manuscript.
Minor concerns:
1) The authors should shorten the words of the Keywords section through stress the main point of the manuscript.
2) For the title of the manuscript, the authors may need to revise it as such title cannot present the manuscript.
3) All the pictures of the manuscript in each Figure reads like screenshotted. The authors should provide pictures with high resolution as accurate as possible. For example, the top of Figure 1 contains a little black letter. What is more, Figure 2 should be redrawn as general readers cannot catch the meaning of the picture. For Figure 3 to Figure 9 and Figure 11 to Figure 18, grey top and right lines were read and such lines should be deleted.
4) For the whole manuscript, the reference especially the number of each reference in the main body of the manuscript should be checked. For example, line 125 and line 160 (reference 7), line 200 (reference 8).
Author Response
1) The authors introduce the concept of vPCR to estimate target copy numbers. While this is an interesting theoretical idea, its practical use is not very clear. Please add more discussion on the limitations of vPCR in real-world settings. Can a normal laboratory use this method easily? What specific equipment or conditions are required to make vPCR work reliably?
Good suggestion. A last paragraph added to (new) section 4
2) The "extreme" standard curve in the paper is built using purified synthetic DNA. However, real clinical or environmental samples often contain inhibitors that affect PCR performance. Please add a section or some examples discussing how complex sample matrices (like blood or tissue extracts) impact the LOD, LOQ, and the overall shape of the standard curve.
Good suggestion. A last paragraph added to (new) section 3.4.
3) Even though I applaud the authors for vPCR. However, the authors seem like not influent in English writing. Therefore, I recommend the authors polish the whole manuscript.
Our native British English speaker has gone through the manuscript with focus on language.
Minor concerns:
1) The authors should shorten the words of the Keywords section through stress the main point of the manuscript.
Done
2) For the title of the manuscript, the authors may need to revise it as such title cannot present the manuscript.
Done
3) All the pictures of the manuscript in each Figure reads like screenshotted. The authors should provide pictures with high resolution as accurate as possible. For example, the top of Figure 1 contains a little black letter. What is more, Figure 2 should be redrawn as general readers cannot catch the meaning of the picture. For Figure 3 to Figure 9 and Figure 11 to Figure 18, grey top and right lines were read and such lines should be deleted.
Amended
4) For the whole manuscript, the reference especially the number of each reference in the main body of the manuscript should be checked. For example, line 125 and line 160 (reference 7), line 200 (reference 8).
Checked!
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled “The qPCR standard curve” presents a timely and pedagogically valuable tutorial that systematically explains the theory, construction, validation, and interpretation of quantitative PCR (qPCR) standard curves. The article is particularly well-suited for early-career researchers, graduate students, and laboratory technicians who are beginning their journey in molecular diagnostics or gene expression analysis. By distinguishing between the dual purposes of standard curves—assay validation versus sample quantification—the authors address a common source of confusion in routine practice and provide much-needed conceptual clarity.
Minor remarks:
1. lines 427, 432 - "Error! Bookmark not defined" - obvious reference formatting errors. Line 407 "...in Figure 12. Standard samples..." - should be "In Figure 12, standard samples", and some more grammar mistakes here and there. Intensive proofreading recommended.
2. ...and I believe Figure 13 must be mentioned in text as Figure 12, same goes for Figure 14 (mentioned as 13).
3. The term “sampling” is used inconsistently—sometimes referring to aliquoting a sample into a reaction well, other times implying biological sampling. Clarifying this distinction or using "aliquota", "sample", "replicates" would prevent confusion.
Overall - I would reccomend publishing this tutorial after an extensive proofreading.
Author Response
1. lines 427, 432 - "Error! Bookmark not defined" - obvious reference formatting errors. Line 407 "...in Figure 12. Standard samples..." - should be "In Figure 12, standard samples", and some more grammar mistakes here and there. Intensive proofreading recommended.
Manuscript proofread and language checked by our native British English coauthor.
2. ...and I believe Figure 13 must be mentioned in text as Figure 12, same goes for Figure 14 (mentioned as 13).
Amended
3. The term “sampling” is used inconsistently—sometimes referring to aliquoting a sample into a reaction well, other times implying biological sampling. Clarifying this distinction or using "aliquota", "sample", "replicates" would prevent confusion.
Good point - Clarified
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has been significantly improved according to the reviewers' comments
Reviewer 2 Report
Comments and Suggestions for AuthorsThe 2nd edition of the manuscript has been revised better than the 1st edition. The present version is recommended to be accepted by the journal IJMS now. Congratulations to the authors.
