key: cord-0853661-lchxtppi
authors: Bansal, Raman; Mamidala, Praveen; Mian, M. A. Rouf; Mittapalli, Omprakash; Michel, Andy P.
title: Validation of Reference Genes for Gene Expression Studies in Aphis glycines (Hemiptera: Aphididae)
date: 2012-08-01
journal: J Econ Entomol
DOI: 10.1603/ec12095
sha: 9e1636fb626b72f4ba9eff80a9cec3b97f8de8c6
doc_id: 853661
cord_uid: lchxtppi

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-1α; 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.

Quantitative real-time PCR (qRT-PCR) has transformed genetic research (gene expression analysis and molecular diagnostics) because of its rapidity, sensitivity, and reproducibility in quantifying mRNA transcripts (Wong and Medrano 2005 , Espy et al. 2006 , Bustin 2010 . The ability of detecting transcripts expressed at low levels by qRT-PCR has made it a standard protocol for gene expression analysis, thus replacing the conventional mRNA quantiÞcation methods (e.g., Northern blot analysis, competitive RT-PCR, RNase protection assay, and microarrays) (Vandesompele et al. 2002, Klie and Debener 2011) .

Given the sensitivity and reproducibility of qRT-PCR technique, a suitable housekeeping gene (HKG) is a prerequisite for accurate quantiÞcation of mRNA transcripts at a given condition (Vandesompele et al. 2002) . Also referred as reference genes or internal control genes, HKGs are thought to be expressed constitutively across different physiological conditions because they are involved in basic functions of a cell (Butte et al. 2001 ). Because of their inherent property of stable expression across different treatments, various tissues, and developmental stages, HKGs are widely accepted as internal controls in mRNA quan-tiÞcation studies (Bustin 2010). However, several normalization studies using qRT-PCR have demonstrated the variability of HKG expression (Vandesompele et al. 2002, Klie and Debener 2011) , supporting the hypothesis that there is no universal reference gene for all biological systems (Gutierrez et al. 2008) . Although a few HKGs have been reported in insects, a species-speciÞc HKG is recommended for precise mRNA quantiÞcation (Scharlaken et al. 2008 , de Boer et al. 2009 , Hiel et al. 2009 , Hornakova et al. 2010 , Jiang et al. 2010 , Lord et al. 2010 .

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), a native of Asia, has emerged as a serious invasive pest in North America and has caused huge losses to soybean, Glycine max (L.) Merr., production (Heimpel et al. 2010 , Tilmon et al. 2011 cost and evolution of virulent biotypes, respectively (Kim et al. 2008 , Song and Swinton 2009 , Hill et al. 2010 , Tilmon et al. 2011 

cDNA Library Construction and Sequencing. The cDNA library from A. glycines adults was constructed using cDNA Synthesis System kit (Roche Applied Science, Indianapolis, IN) and was sequenced on 454 GS Titanium platform. More details on library construction, sequencing, and data analysis are provided previously (Bai et al. 2010 ). The cDNA library of A. glycines contained 19,293 high-quality transcripts in total. Homology searches for transcript sequences were performed using Blast2GO software [E-value cut-off 10 Ϫ3 ] (Conesa et al. 2005, Conesa and Gö tz et al. 2008 Gö tz et al. , 2011 . Based on information available on commonly used reference genes in the literature, we selected cDNA contigs of various candidate reference genes for A. glycines (Table 1 ). The identity of putative cDNAs was further conÞrmed by Blastx search in GenBank (National Center for Biotechnology Information, Bethesda, MD). The cDNA sequences of candidate reference genes were deposited in GenBank (for accession numbers, see Table 1 ).

Insect Culture. For all qRT-PCR experiments, A. glycines insects were obtained from a laboratory colony, referred as biotype 1 (B1) that originated from insects collected from Urbana, IL (40Њ 06Ј N, 88Њ 12Ј W) in 2000 (Hill et al. 2004) . At Ohio Agricultural Research and Development Center (Wooster, OH), a laboratory population of these insects is maintained on susceptible soybean seedlings [SD01-76R (2)] in a rearing room at 23Ð25ЊC and a photoperiod of 15:9 (L:D) h.

Freshly hatched Þrst-instar nymphs of A. glycines (60 Ð70 individuals) were allowed to feed on resistant [LD-05 16060, contains Rag1] and susceptible soybean [SD01-76R (2)] plants (Tinsley et al. 2012 ). After 12 h of feeding, insects were collected and placed in Ϫ80ЊC. There were three biological replications for each treatment. The collected insect samples were processed for total RNA extraction by using TRI reagent (Molecular Research Center Inc, Cincinnati, OH), following the protocol provided by the manufacturer. RNA samples were treated with TURBO DNase (Applied Biosystems/Ambion, Austin, TX) to remove any DNA contamination. Using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA), Þrst strand cDNA was prepared with 500 ng of RNA. qRT-PCR reactions were performed with iQ SYBR Green super mix on a CFX-96 thermocycler system (Bio-Rad Laboratories) (Bansal et al. 2011). SpeciÞc primers for each candidate reference gene were designed using Beacon Designer version 7.0 (Premier Biosoft, Palo Alto, CA) ( Table 2) . Each reaction was performed with 1 l of cDNA, 0.5 M of each primer, and 12.5 l of iQ SYBR Green super mix in a 25-l total volume. Each reaction was done in duplicate in a 96-well optical-grade PCR plates, sealed with optical sealing tape (Bio-Rad Laboratories). The PCR ampliÞcations were done with the following cycling conditions: one cycle at 95ЊC (3 min), followed by 35 cycles of denaturation at 95ЊC (30 s), annealing and extension at 55ЊC for 45 s. Finally, melt curve analyses were done by slowly heating the PCR mix- 

To obtain selected tissue samples (gut, fat body, integument, and embryo developing inside adults), A. glycines adults (5 d old) were dissected out in phosphate-buffered saline, pH 8.0, under a dissecting microscope. During dissection, other tissues of A. glycines such as salivary glands and bacteriocytes were discarded. To determine the expression of candidate reference genes in different developmental stages, all the four nymphal and adult samples were collected from insects feeding on susceptible soybean [SD01-76R (2)] plants. Both tissue and developmental stagesÕ samples were processed for total RNA extraction, DNase treatment, Þrst strand cDNA synthesis, and qRT-PCR as described in the previous section. There were two biological and two technical replications for each. The Þrst strand cDNA was prepared with 150 and 500 ng of RNA (DNA free) from tissue and developmental stages samples, respectively.

Stability Analysis of Candidate Reference Genes. Two software algorithms, i.e., GeNorm (Vandesompele et al. 2002) and NormÞnder (Andersen et al. 2004 ) were used to determine the stability of candidate reference genes. The raw expression values of each gene, calculated by equation 2 (Ϫ⌬Ct) were used as input data for both GeNorm and NormÞnder. GeNorm calculated the M-score, and the lower the value for M is indicative of a more stable expression or low variation (Vandesompele et al. 2002) . This value is calculated by a geometric averaging of all the reference genes used in the study and mean pairwise variation of a reference gene from other reference genes. It is important to note that the HKGs showing higher M value (M Ͼ 1.5) are not considered for normalization studies. NormFinder also determines the expression stability but by taking account of intraand intergroup variations for candidate reference genes (Andersen et al. 2004) . NormFinder provides the stability value for each gene, a direct measure for the estimated expression variation enabling evaluation of the systematic error introduced when using the gene for normalization (Andersen et al. 2004) .

We proÞled seven commonly used HKGs (SDFS, EF1a, HEL, GAPDH, RPS9, TBP and UBQ) from a transcriptomic database of A. glycines. The preliminary screening of these HKGs presented a range of Ct values across the treatments (16.87Ð26.11, A. glycines fed with resistant and susceptible host plants), tissues (21.52Ð31.33, epidermis, gut, fat body, and embryo), and developmental stages (17.08 Ð29.15, ÞrstÐfourthinstar nymphs and adults) of A. glycines (Fig. 1) . Our qRT-PCR analysis was highly optimized as ampliÞcation efÞciencies (E) for various primer pairs ranged from 90.44 to 108.16 and R 2 were Ͼ0.94 (Table 2 ) (Schmittgen and Livak 2008) . We included the melting curve analysis (65Ð95ЊC in increments of 0.5ЊC every 5 s) in qRT-PCR reaction that revealed the lack of any nonspeciÞc product ampliÞcation (Supp. Fig. 1 [online only]). To determine a suitable reference gene for gene expression studies in A. glycines, we used GeNorm (version 3.5) and NormFinder that are freely available software (Vandesompele et al. 2002 , Andersen et al. 2004 .

GeNorm Analysis. Among the seven reference genes (SDFS, EF1a, HEL, GAPDH, RPS9, TBP, and UBQ), GeNorm revealed TBP to be stably expressed across the treatments, tissues, and developmental stages (Fig. 2AÐC, respectively) . Although EF1a showed stable expression in both tissues and developmental stages, it had the least stability in insects fed on resistant and susceptible host plants. GeNorm determined the gene stability measure (M) among all the reference genes tested. Except for the EF1a in treatments, all the HKGs assayed in the current study displayed M Ͻ 1.5. This trend of dissimilar ranking of reference genes across treatments, tissues, and developmental stages is quite a common phenomenon observed in several gene expression studies in insects (Hiel et al. 2009 , Hornakova et al. 2010 al. 2011) . Hence, it is highly recommended to determine the stably expressed reference gene for a given sample as normalization is critical and a significant component of Þnal data interpretation.

Although a single reference gene which is stable and highly expressed sufÞces the requirement of quantifying mRNA transcript levels for a gene of interest, it also is recommended to use at least two to three reference genes for effective normalization of gene expression data (Vandesompele et al. 2002) . The requirement of optimal number of HKGs can be obtained from the pairwise variation (V), wherein Vandosomplele et al. (2002) proposed a cut-off value (0.15), below which the inclusion of other reference genes are not required. Results in the current study showed that the application of at least two stable reference genes maintains proper normalization irrespective of treatment, tissue, and development stagesÕ samples (Fig. 3AÐC, respectively) . Because the pairwise variation was calculated by adding candidate genes stepwise according to rankings shown in Fig. 2 , two of the most stable genes identiÞed for a particular experimental condition (Fig. 2) should be able to be combined for normalization. The candidate genes having lower stability in a given treatment (Fig. 2) should be avoided for normalization even if it is to be used in combination with other reference gene given the suitability and availability of other reference genes. For example, GeNorm analysis suggests that for qRT-PCR normalization in A. glycines fed with resistant and susceptible soybean, both TBP and RPS9 could be used together. Similarly, for normalization among different developmental stages and among tissues of A. glycines, TBP and EF1a could be combined. It is interesting to note that various HKGs (RPS9, GAPDH, HEL, and UBQ) were not consistently ranked in terms of their stability under different experimental conditions, that is also the case observed in other insect studies (Hiel et al. 2009 , Hornakova et al. 2010 , Jiang et al. 2010 , Lord et al. 2010 ). Therefore, care should be taken in determining which HKG to use depending on experimental conditions. NormFinder Analysis. NormFinder showed similar results to GeNorm, wherein TBP was shown to have low stability value (lower variation of gene expression) across all the samples (Fig. 4) , further indicating the potential for this gene as a HKG for gene expression studies in A. glycines. Although there are discrep- ancies between the GeNorm and NormÞnder output, TBP was shown to be the best ranking reference gene with both methods among treatments, tissues, and developmental stages ( Fig. 2 and 4) . Also, both of these analyses displayed RPS9 as an appropriate HKG across the treatments ( Figs. 2A and 4A) . The different ranking of other HKGs (EF1a, GAPDH, HEL, and UBQ) by both GeNorm and NormFinder clearly demonstrates the expression stability of HKGs is inßuenced spatially, temporally, and also on experimental treatment.

In conclusion, the HKG gene TBP showed stable expression across all the samples and did not seem to be inßuenced by treatments (A. glycines fed with resistant and susceptible host plants), among the various tissues and developmental stages included. Therefore, it could be used to normalize the mRNA transcript levels of candidate genes in A. glycines. The TBP is a transcription factor that binds speciÞcally to a DNA sequence called the TATA box and is well documented for its use as reference gene in several studies (Radonic et al. 2005 , Jung et al. 2007 , Nygard et al. 2007 ). In addition, for experiments with A. glycines fed with resistant and susceptible host plants, RPS9 may be considered. Similarly, for normalization among tissues and developmental stages, EF1a also can be used along with TBP. The identiÞed reference genes in the current study may potentially serve as ideal internal controls in other closely related aphid species. However, the order of the gene stability should be revised for using a species speciÞc study.

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This work was supported by a grant from North Central Soybean Research Program and The Ohio Soybean Council (11-2-04). The CFX-96 thermocycler system was purchased, in part, by an equipment grant from Department of Entomology and Ohio Agricultural Research and Development Center.

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