title: Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae).
abstract: Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-la; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant-resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines.
authors.affiliation: Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA.
authors.forename: Raman
authors.initials: R
authors.lastname: Bansal
authors.forename: Praveen
authors.initials: P
authors.lastname: Mamidala
authors.forename: M A Rouf
authors.initials: MA
authors.lastname: Mian
authors.forename: Omprakash
authors.initials: O
authors.lastname: Mittapalli
authors.forename: Andy P
authors.initials: AP
authors.lastname: Michel
mesh_terms: D000818:Animals; D001042:Aphids / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020043:Genes, Essential; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D020648:Peptide Elongation Factor 1; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; D035181:TATA-Box Binding Protein; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D044763:Ubiquitin-Conjugating Enzymes