Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg
Round 1
Reviewer 1 Report
Minor corrections: Units should be checked in the M&M (eg. lane 245 the unit is l instead µl.
Lanes 248-250 are repeated
Author Response
1、Minor corrections: Units should be checked in the M&M (eg. lane 245 the unit is l instead µl.
Lanes 248-250 are repeated.
1、We have deleted the repeated sentence. And we corrected the structure of sentence, and added losing symbol to correct location.
Reviewer 2 Report
This study sets out to identify useful reference genes for Liriodendron chinense. The choice of good reference genes is extremely important for RT-qPCR, but is still often neglected. Thus, this is a useful study that will help scientist working with this species. The verification with a specific target gene was less clear to me, as it can only be called a verification if the expression level of this target gene was already known.
Comments:
INTRODUCTION:
“If the expression level of the reference gene has a very large experimental error, then the noise of the assay will be large, and small changes in the expression of target genes will be impossible to detect.”
This study is not looking into the “experimental error” and resulting “noise”, but rather into “expression stability” and resulting normalization; restate.
“In addition, to verify our study results, the LcPAT7 (Liriodendron chinese protein S-acyltransferase 7) gene was chosen as the target gene, and its expression level was determined by RT-qPCR analysis.”
Was the expression of this gene known? If not, how could it verify the results? Clarify (also in results and discussion)
RESULTS
Figure 2 and 3: figure legends are difficult to read (too small)
When mentioning concepts such as average expression stability (M) or pairwise variation (Vn/n+1) for the first time in the text, briefly explain the concept.
DISCUSSION
“However, no report on selecting suitable reference genes systematically for RT-qPCR analysis in L. chinense .” incomplete sentence
MATERIALS AND METHODS
the symbols for “micro” and “degree” got lost throughout the text
Author Response
1、This study is not looking into the “experimental error” and resulting “noise”, but rather into “expression stability” and resulting normalization; restate.
1、This sentence is changed to the following sentence: If the expression pattern of the reference gene is unstable, then the accuracy of the assay will be reduced, and small changes in the expression of the target genes will be impossible to detect.
2、Was the expression of this gene known? If not, how could it verify the results? Clarify (also in results and discussion)
2、The expression of this gene was unknown in the S3 state, so we changed the way we describe into comparison of the differences between reference genes we selected and unsuitable candidate reference genes when using as internal control for LcPAT7 RT- PCR analysis (lane 199, 280).
3、Figure 2 and 3: figure legends are difficult to read (too small)
3、Image size has changed into bigger.(Fig.2 and 3)
4、When mentioning concepts such as average expression stability (M) or pairwise variation (Vn/n+1) for the first time in the text, briefly explain the concept.
4、We have added the concept of average expression stability (M) or pairwise variation (Vn/n+1) to the article.(lane 105 to 110).
5、“However, no report on selecting suitable reference genes systematically for RT-qPCR analysis in L. chinense .” incomplete sentence.
5、This sentence is changed to the following sentence: However, no report exist of systematically selecting suitable reference genes for RT-qPCR analysis in L. chinense. Therefore, suitable internal control genes for RT-qPCR analysis must be carefully identified in L. chinense.
6、the symbols for “micro” and “degree” got lost throughout the text
6、We have added the the symbols for “micro” and “degree” to the text.(lane lane 60-61, lane 80, lane 93-100)
Reviewer 3 Report
This study identified the reference genes to improve qRT-PCR based gene expression analysis in L. chinense. Line 41-42 says "However, little 42 molecular and gene expression data have been reported for L. chinense. A". This statements points towards a very limited scope of this study. I was not convinced enough about the substantial need for doing such a work, unless the tree organs used in this study are of high importance.
Although the study provides a useful set of genes with stable expression across various tissues, I feel like the sample size is quite limited for their utility. Only leaf and flower tissues were used at different developmental stages. There was a mention of root and twigs, but it seems like the samples were collected from them at only single time point. This kind of work should extend beyond a limited sample size.
Overall, this study has a limited merit and scope, or the introduction part was not convincing enough to describe it clearly. I recommend revising the introduction by improving scope and importance of this work in L. chinense and related species. The study can widen the tissues used for expression analysis, and also if possible to perform such an analysis under most important bitoic and abiotic stresses for L. chinense.
Author Response
1、This study identified the reference genes to improve qRT-PCR based gene expression analysis in L. chinense. Line 41-42 says "However, little 42 molecular and gene expression data have been reported for L. chinense. A". This statements points towards a very limited scope of this study. I was not convinced enough about the substantial need for doing such a work, unless the tree organs used in this study are of high importance.
1、The organs we selected are important, because most studies about L. chinense are focus on leaf and flowers. Such as, leaves were used as materials for L. chinense sequencing and its genome assembling had been finished in 2018 (Jinhui Chen, Zhaodong Hao, Xuanmin Guang et al., Liriodendron genome sheds light on angiosperm phylogeny and species–pair differentiation. NAT. PLANT, 2018, https://doi.org/10.1038/s41477-018-0323-6.), Kangqin Li et al. used leaves as materials to study the genetic diversity of L. chinense (Kangqin Li, Long Chen, Yuanheng Feng et al., High genetic diversity but limited gene flow among remnant and fragmented natural populations of Liriodendron chinense Sarg, BIOCHEM. SYST. ECOL, 2014, http://dx.doi.org/10.1016/j.bse.2014.01.019), Ying Yang et al. used petals and leaves as materials to study the transcriptome of L. chinense (Ying Yang, Meng Xu, Qunfeng Luo et al., De novo transcriptome analysis of Liriodendron chinense petals and leaves by Illumina sequencing, Gene, 2014,
http://dx.doi.org/10.1016/j.gene.2013.10.073). Due to genome sequencing of L. chinense had been finished, it would facilitate the study of functional genes. However, no one systematically selected the reference genes, it will influence the accuracy of RT-qPCR analysis. Therefore, the expression of functional genes cannot be characterized.
2、Although the study provides a useful set of genes with stable expression across various tissues, I feel like the sample size is quite limited for their utility. Only leaf and flower tissues were used at different developmental stages. There was a mention of root and twigs, but it seems like the samples were collected from them at only single time point. This kind of work should extend beyond a limited sample size.
2、Although the number of samples in this study was only 54 and the samples were mainly leaves and flowers, the current study mainly focused on flowers and leaves. The purpose of this study was to meet the current needs, so we only used 54 materials.
3、Overall, this study has a limited merit and scope, or the introduction part was not convincing enough to describe it clearly. I recommend revising the introduction by improving scope and importance of this work in L. chinense and related species. The study can widen the tissues used for expression analysis, and also if possible to perform such an analysis under most important bitoic and abiotic stresses for L. chinense.
3、The habitat of L. chinense is relatively mild, and L. chinense is rarely affected by biotic and abiotic stress in its natural state. On the other hand, reference genes have species specificity and tissue specificity, these limit its application category. So, according to the practical situation, we only selected reference genes for L. chinense and its vegetative organs and flower organs as materials. And our research results have been put into practice ( not publish).
