Determination of Suitable RT-qPCR Reference Genes for Studies of Gene Functions in Laodelphax striatellus (Fallén)

Abstract

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Lsα-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Lsβ-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.

Keywords: Laodelphax striatellus; RT-qPCR; reference gene; rice black-streaked dwarf virus; rice stripe virus.

Figure 1

Ranges of expression of the seven selected reference genes in all analyzed samples. The whisker box plots represent the cycle threshold numbers (Ct value) of the seven reference genes. The horizontal lines inside the boxes are the median values, and the whiskers of the boxes are the minimum and maximum Ct values.

Figure 2

Rankings of reference gene expression stabilities determined by RT-qPCR followed by geNorm, Bestkeeper, Normfinder, or the RefFinder algorithm. Results represent individual reference genes. (A–D) L. striatellus at all developmental stages; (E–H) tissues from non-viruliferous (NV) L. striatellus; (I–L) tissues from Rice black-streaked dwarf virus viruliferous (RBSDV-V) or NV L. striatellus; (M–P) RBSDV-V or Rice stripe virus viruliferous (RSV-V) L. striatellus; (Q–T): RBSDV-V and Lssynaptojanin 1 (LsSYNJ1) -silenced L. striatellus, or RBSDV-NV and LsSYNJ1-silenced L. striatellus.

Figure 3

Pairwise variations were calculated using the geNorm algorithm to determine the optimal numbers of reference genes needed for accurate RT-qPCR data normalization. The pairwise variation (V_n/V_n+1) was calculated using data from different developmental stages, different tissues, RBSDV-NV, RBSDV-V, or RSV-V L. striatellus, LsSYNJ1-silenced L. striatellus, and L. striatellus in all tested samples.

Figure 4

Determinations of RBSDV P5-1 and RBSDV P10 expression using different reference genes. (A,B) The RT-q PCR data were normalized using most suitable reference gene Lsα-TUB or the least stable reference gene LsRPL8 in different tissues including the head (black), cuticle (light gray), fat body (white), and midgut (gray). (C,D) The RT-q PCR data were normalized using most suitable reference gene Lsβ-TUB and least stable reference gene LsGAPDH in dsGFP (gray) and LsSYNJ1-silenced samples (black). **means p < 0.005, *** means p < 0.001.