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, 14 (8), e0220475
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Selection of Reference Genes for Expression Analysis of Plant-Derived microRNAs in Plutella Xylostella Using qRT-PCR and ddPCR

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Selection of Reference Genes for Expression Analysis of Plant-Derived microRNAs in Plutella Xylostella Using qRT-PCR and ddPCR

Lingling Zhang et al. PLoS One.

Abstract

The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-derived miRNAs, which paves the way for unveiling their roles in the insect-plant coevolution.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cycle threshold (Ct) values of candidate reference genes.
The Ct values of the candidate reference genes were measured in the samples from G88 and GC strains as described in the Materials and Methods.
Fig 2
Fig 2. Stability analysis of candidate reference genes in the developmental stages of G88 strain.
The Ct values derived from samples including first-day eggs, 1st instar larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, pupae and adults were used to calculate the stability values by different algorithms. A lower value indicates a more stable candidate gene as reference gene (numbers reported above the columns).
Fig 3
Fig 3. Stability analysis of candidate reference genes in the tissues of G88 strain.
The Ct values derived from samples including brain, midgut, silk gland, Malpighian tubule, fat body, hemolymph, and the remaining tissues of 4th instar larvae were used to calculate the stability values by different algorithms. A lower value indicates a more stable candidate gene as reference gene (numbers reported above the columns).
Fig 4
Fig 4. Stability analysis of the candidate reference genes in the tissues of GC strain.
The Ct values derived from samples including midgut, silk gland, Malpighian tubule, fat body, hemolymph and remaining tissues from the first-day 4th instar larvae were used to calculate the stability values by different algorithms. A lower value indicates a more stable candidate gene as reference gene (numbers reported above the columns).
Fig 5
Fig 5. Determination of the optimal number of reference genes for normalization.
(A) Developmental stages of G88 strain; (B) Tissues of G88 strain; (C) Tissues of GC strain. Pairwise variations (Vn/n+1) were calculated between the normalization factors NFn and NFn+1 by geNorm to determine the optimal number of reference genes. geNorm decides whether inclusion of an extra reference gene adds to the stability of the normalization factor.
Fig 6
Fig 6. Validation of reference gene selection using qRT-PCR.
Relative expression levels of the target gene ath-miR159a in the tissue samples of the GC strain were measured by four normalization factors. NF1, normalized against the best reference gene; NF(1–2), normalized against the two most stable reference genes; NF(1–3), normalized against the three most stable reference genes; and NF4, normalized against the least stable reference gene. MG: midgut, SG: silk gland, MT: Malpighian tubule, FB: fat body, HE: hemolymph, RE: remaining tissues. Expression levels were independently compared among tissues for each normalization factor. The data were presented as the mean ± SD with level in midgut normalized to 1 (one-way ANOVA followed by a Tukey’s multiple comparison test, p < 0.05).
Fig 7
Fig 7. Validation of reference gene selection using ddPCR.
MG: midgut, SG: silk gland, MT: Malpighian tubule, FB: fat body, HE: hemolymph, RE: remaining tissues. The data were presented as the mean ± SD (one-way ANOVA followed by a Tukey’s multiple comparison test, p < 0.05).

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Grant support

This study was funded by the National Key R&D Program of China (grant number 2017YFD0200400 awarded to Assoc. Prof. Weiyi He), the Fujian Provincial Science and Technology Major Project (grant number 2018NZ0002-1 awarded to Prof. Minsheng You), and the Natural Science Foundation of Fujian Province (grant number 2019J01369 awarded to Assoc. Prof. Weiyi He). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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