Selection of Reference Genes for Expression Analysis Using Quantitative Real-Time PCR in the Pea Aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae)

Abstract

To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin), elongation factor 1 α (EF1A), TATA-box-binding protein (TATA), ribosomal protein L12 (RPL12), β-tubulin (Tubulin), NADH dehydrogenase (NADH), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase B (SDHB), 28S ribosomal RNA (28S), 16S ribosomal RNA (16S), and 18S ribosomal RNA (18S) from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.

Evaluation of the reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the green peach aphid, Myzus persicae

“…The rankings of candidate genes differ among various treatments based on the different algorithms and different sensitivities toward co‐regulated reference genes (Fu et al ., , Yang et al ., ). BestKeeper analyzes individually the stability of reference genes.…”

“…Thus, a stable reference gene is an important prerequisite in any experiment. Currently, the selection of reliable reference genes have been accomplished for the diamondback moth, Plutella xylostella L. (Fu et al ., ), the sweetpotato whitefly, Bemisia tabaci (Gennedius), the brown planthopper, Nilaparvata lugens L. (Yuan et al ., ), the pink stem borer, Sesamia inferens Walker (Sun et al ., ), the honey bee (Scharlaken et al ., ), the locust (Van Hiel et al ., ; Chapuis et al ., ), the psocid, Liposcelis bostsrychophila Badonnel (Jiang et al ., ), the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Shi et al ., ), the oriental fruit fly, Bactrocera dorsali s (Hendel) (Shen et al ., ), Drosophila melanogaster L. (Ponton et al ., ), the cotton bollworm, Helicoverpa armigera (Hübner) (Zhang et al ., ) and the pea aphid, Acyrthosiphon pisum (Harris) (Yang et al ., ). In this work, we used five analysis systems to evaluate the suitability of the nine candidate reference genes under various conditions.…”

Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants

“…Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with previously published methods (Yang et al, 2014, 2015). DNase treated total RNA was denatured at 75°C for 5 min and immediately chilled on ice.…”

“…Details regarding RT-qPCR amplifications and programs were provided in Yang et al (2014); briefly, PCR reactions (20 μl) contained 7.0 μl of ddH 2 O, 10.0 μl of 2× SYBR Green Master Mix (Bio-Rad), 1.0 μl of each specific primer (10 μM), and 1.0 μl of first-strand cDNA template. Reactions occurred in 96-well format Microseal PCR plates (Bio-Rad) in triplicate.…”

Evaluation of a cuticle protein gene as a potential RNAi target in aphids

“…EF1α and ribosomal protein S20 ( RPS20 ) were used to normalize the expression of Ac. pisum CP19 ( ApCP19 ), and Actin and para were used to normalize the expression of M. persicae CP19 ( MpCP19 ) . 18S ribosomal RNA ( 18S ) and β‐act were used to normalize the expression level of P. japonica CP19 ( PjCP19 ) …”