Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae)

Abstract

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance.

Keywords: Mylabris sibirica; RT-qPCR; RefFinder; geNorm; reference gene.

FIGURE 1

Expression levels of ten house-keeping genes in Mylabris sibirica. The mean C_t values for 10 candidate reference genes are shown in four different experiments: (A) adult ages, (B) adult tissues, (C) temperature, (D) sex. Mean Ct values for the eight candidate reference genes are presented in box plots, where each box indicates the 25th and 75th percentiles, and the line across the box represents the median. Abbreviation: ACT, actin; ARF1, ADP ribosylation factor 1; AK, arginine kinase; α-TUB, α-tubulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EF1α, elongation factor 1α; RPL6, RPL13, RPS3 and RPS18, ribosomal protein. The abbreviations are exactly the same as Figure 2 through Figure 6.

FIGURE 2

Stability of the ten house-keeping genes in Mylabris sibirica during diverse adult ages. All stages of Mylabris sibirica adults were sampled (collected on the first to third days of the newly-emerged adults). The expression stability rankings are determined by geNorm, NormFinder and BestKeeper.

FIGURE 3

Stabilities of the ten house-keeping genes in Mylabris sibirica among various adult tissues. Foregut, midgut, hindgut, epidermis, trachea and antenna were dissected from the newly-emerged adults. The expression stability rankings are determined by geNorm, NormFinder and BestKeeper.

FIGURE 4

Stability of the ten house-keeping genes in Mylabris sibirica under different temperatures. The newly-emerged adults reared under five temperatures (4°C, 15°C, 20°C, 26 °C and 35 °C) were collected. The expression stability rankings are determined by geNorm, NormFinder and BestKeeper.

FIGURE 5

Stabilities of the ten house-keeping genes in Mylabris sibirica in males and females. The male and female adults were collected at day 5 after emerging. The expression stability rankings are determined by geNorm, NormFinder and BestKeeper.

FIGURE 6

Stabilities of the ten house-keeping genes in Mylabris sibirica in different samples. The stability of the reference genes as calculated by the Geomean method of RefFinder. A lower Geomean of ranking value denotes more stable expression. (A) Adult ages, (B) adult tissues, (C) temperature, (D) sex, (E) all samples.

FIGURE 7

Relative gene expression of UAP in different adult tissues of Mylabris sibirica. The relative gene expression level of UAP in the foregut, midgut, hindgut, epidermis, trachea and antenna was normalized to the best stable [(A) RPL6 and RPS18] and least stable [(B) AK and GAPDH] reference genes, respectively. The values are means +SE. Different letters indicate significant differences in gene expression among different tissues (p < 0.05).