Selection and Identification of a Reference Gene for Normalizing Real-Time PCR in Mangos under Various Stimuli in Different Tissues

Round 1

Reviewer 1 Report

Dear authors, identify appropriated reference genes for gene expression analysis is an important field for molecular biology. As this kind of research impacts strongly in the obtained results in gene expression assays, the methods used to identify stable reference genes must be clear and the reader can critically evaluate if the recommended reference genes are suitable or not for your work. It was not offer to me the supplementary data so I couldn’t have access to the entire data of the article. Based on that, I listed below some points mainly related to material and methods.

Line 80: The application of the treatments need more information to be clear for the reader. First, the abiotic stresses treatments were performed only in leaves? For root, stem and fruit samples did not receive any abiotic stress treatment? Also, how the abiotic stress treatment was applied? Do you first collected the leaves and after applied the treatment? Do you have a reference for this method? It is strange since as you quantifying specifically the transcript number of the reference genes, when the leaves are collected (before the application of the treatments) you are affecting the cell homeostasis and consequently the gene expression.

Line 95: Which kind of oligonucleotide was used to convert RNA into cDNA? It is important to inform it because the reader should know the strategy used to synthesized the cDNA and infer in your results.

Line 101: Most of your amplicons in table 1 are higher than 150 pb. It makes me think about the 30 seconds extension. The DNA polymerase used in RT-qPCR analysis is able to amplify amplicons higher than 150 pb in 30 seconds? Also, you have to change that information related the amplicon size.

Line 102: Taking into account the size of your amplicon, how could you verify specific amplified products in a 1% agarose gels? Also, which kind of PCR you used to access the oligonucleotide efficiency? How was the constitution of your PCR reactions for efficiency analysis?

In addition, what about the melting curves? How did you verify the specificity of your oligonucleotides? How many dilutions points were used for standard curves?

Line 103: The use of "ordinary PCR" makes no sense for that kind of analysis. First because test oligonucleotides in one kind of thermocycler and project it for other kind of thermocycler is unusual; second because you can't infer in RT-qPCR (very small amplicons) oligonucleotide specificity by agarose gel; and finally, when you perform your RT-qPCR assay you have a very sensitive analysis offering you melting curves that gives you the specificity of your oligonucleotide.

Line 112: Are you sure you used only 5 seconds for initial denaturation?

Line 113: I suspect that you used a SYBR mix containing the DNA polymerase, co-factors, dNTPs, etc. Please let it clear in your text.

Table 1: The TM are too low for RT-qPCR analysis since the amplicon is too small there is a chance of unspecificity. I suggest to provide the melting graphics of all the oligonucleotides.

Figure S1: I have not access to that file, and it is important because I can't realize that unspecificity/specificity for an oligonucleotide designed for RT-qPCR can be visible in 1% agarose gels due its amplicon size that is frequently together with the rest of primer or primer dimer in the end of the gel. 1% agarose gels are to low concentrated and I guess you could not see the specificity of your RT-qPCR oligonucleotides either in 2% agarose gels. Thus, as a paper describing reference genes for RT-qPCR I strong suggest to show the melting curves of your oligonucleotides as specificity test.

Line 165: Are you sure about the Ct value of 18s? It is a gene that is highly expressed but I never saw that Ct value too early for 18s.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Work sent to review presents interesting and important results but needs more work of authors in the methods and result description. 

Authors use RT-qPCR and qRT-PCR in the main work, however, real-time PCR is a method for quantitative analysis. Authors should use "Real-Time PCR" or "quantitative PCR" in the main text. I recommend "Real-Time PCR". 

Please describe methods of root, stem, and fruit treatment in materials and methods section.

Why do authors use two kits for RNA extraction? If the first figure presents the mean Ct Value for all samples, extraction using each kit should be present separately, i.e., Ct Value for leaf, and Ct Value for other samples.

How many ug of total RNA was used for DNase I treatment? Concentration was measured after DNase I treatment or before?

For cDNA synthesis, do authors use oligo(dT) or random hexamer primers?

Why did author decided to use a double delta while analysis using a standard curve for copy number of investigated genes is more precise (what will be recommended for the type of this article?

Many places indicate "Error! Reference source not found".

I recommended figure presented consecutive steps of this analysis due o the fact that many analyses were performed using leaves. It will get the information right. 

In Figure 2 on the top should be "stability".

Data are present in supplementary materials starting from "Table S2". I don't see supplementary materials and I can't review them. 

In summarizing, the work presents important results but much detailed information should be improved.

Thank you.

 

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript is suitable in the present form. Hwever, I strongly don't agree with the "Real time PCR" term for the type of assay used. I believe that the "RT-qPCR" reflects the used tool and is a suitable and is in accordance with the literature.

Reviewer 2 Report

Dear Authors,

Thank You for the answer and correct the manuscript. Data present in the main text are very important, however, while we will be selected reference genes for quantitative analysis, we should remember that TUBB can be suitable for this experimental setup however when we use plants growth under stress conditions, cells are smaller than in control and the TUBB or ACT genes expression will be different.

Kind regards