Suitable Internal Reference Genes Research Articles

Evaluation of suitable reference genes for expression profile analyses of target genes in the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae).

The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae), is a major destructive insect pest of coffee, which impacts the coffee crops negatively. As a draft genome has been completed for this insect, most molecular studies on gene transcriptional levels under different experimental conditions will be conducted using real-time reverse-transcription quantitative polymerase chain reactions (RT-qPCR). However, the lack of suitable internal reference genes will affect the accuracy of RT-qPCR results. In this study, the expression stability of nine candidate reference genes was evaluated under different developmental stages, temperature stress, and Beauveria bassiana infection. Data analyses were completed by four commonly used programs, BestKeeper, NormFinder, geNorm, and RefFinder. The result showed that RPL3 and EF1α combination were recommended as the most stable reference genes for developmental stages. EF1α and RPS3a combination were the top two stable reference genes for B. bassiana infection. RPS3a and RPL3 combination performed as the optimal reference genes both in temperature stress and all samples. Our results should provide a good foundation for the expression profile analyses of target genes in the future, especially for molecular studies on insect genetic development, temperature adaptability, and immune mechanism to entomogenous fungi in H. hampei.

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera.

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata.

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai-Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

Cloning the full-length CDNA of actin gene and analysing alliinase gene expression in tillering onion

Tillering onion is a herbaceous plant belonging to the Liliaceae family. We cloned the cDNAs of the actin gene (AcACT, GenBank: MF919598) of tillering onion using rapid amplification of the cDNA ends. The full-length cDNA of AcACT was 1,357 bp long with an open reading frame of 1,131 bp encoding 376 amino acids. The amino acid sequence of AcACT shared > 96% similarity with the amino acid sequences of other ACTs and was found (by means of phylogenetic tree analysis) to be closely related to those of Ananas comosus and Papaver somniferum. AcACT expressions showed no significant differences (p > 0.01) in two cultivars L-SH and L-SY over three growth periods and under suitable conditions, low temperature, and short-day conditions. In addition, AcACT was used as an internal reference gene to analyse the expression of the alliinase gene (AcALL). AcALL expression trends in the roots, stems and leaves were consistent with those of diallyl disulphide and diallyl trisulphide. Thus, AcACT is highly conserved and can be used as a suitable internal reference gene when analysing gene expression in tillering onion.

Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary.

Normalization of the expression profiling of target genes, in a tissue-specific manner and under different experimental conditions, requires stably expressed gene(s) to be used as internal reference(s). However, to study the molecular regulation of oocyte meiosis initiation during ovary development in chicken embryos, stable reference gene(s) still need to be compared and confirmed. Six candidate genes previously used as internal references for the chicken embryo (Actb, Cvh, Dazl, Eef1a, Gapdh and Rpl15) were chosen, and their expression profiles in left ovaries dissected at five chicken embryonic days (E12.5, E15.5, E17.5, E18.5 and E20.5) were evaluated, respectively. Separately, GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods were used to assess the stability of candidate reference genes, and all results were combined to give the final rank by RefFinder. All methods identified that Eef1a and Rpl15 were the two most stable internal reference genes, whereas Cvh is the most unstable one. Moreover, expression levels of three marker genes for chicken oocyte meiosis entry (Stra8, Scp3 and Dmc1) were normalized, based on Eef1a, Rpl15, or their combinations, respectively. Our findings provide the most suitable internal reference genes (Eef1a and Rpl15), to investigate further molecular regulation of ovary development and oocyte meiosis initiation in chicken embryos.

Selection of suitable reference genes for miRNA expression normalization by quantitative RTPCR under abiotic stress in Larix olgensis

Abstract Suitable selection of endogenous controls is necessity for obtaining reliable qRT-PCR results. However, insufficient attention has been paid to the selection of endogenous controls for normalization in different stresses in plant, especially Larix olgensis. In this study, we screened candidate internal reference genes for miRNA expression normalization after subjecting Larix olgensis PEG-induced drought, NaHCO3, or NaCl stress. Needles samples were collected at 0, 12, 24, 48 and 96 h after treatment. Four common housekeeping genes and 11 mature miRNAs that were found to be stably expressed in small RNA sequencing data of Larix olgensis were selected as candidate internal reference genes. Their expression levels were assessed by qRT-PCR. GeNorm and NormFinder were used to analyse the stability of gene expression from the candidate internal reference genes. Finally, the expression of some miRNAs was analysed after stress. The results showed that miRNAs exhibited better expression stability compared to the analysed ncRNAs. Under PEG, NaHCO3, or NaCl stress, miR83321, miR43169, and miR74940 were identified as the most suitable internal reference genes, respectively. Under PEG stress, the expression of most miRNAs reached its highest level at 12 h, showing a trend of first increasing expression and then decreasing. Similarly, under NaHCO3 and NaCl stress, the expression of most miRNAs reached its maximum levels at 48 h and 12 h, respectively. This indicates that most miRNAs more quickly respond to PEG and NaCl stress than NaHCO3 stress.

The identification of suitable internal reference genes in quinoa seeds subjected to abscisic acid and gibberellin treatment

Abstract: Quinoa has been recognized as the sole “comprehensive nutritional crop”; however, it is susceptible to pre-harvest sprouting (PHS). While quantitative reverse transcription polymerase chain reaction (RT-qPCR) has been extensively employed for gene expression level detection, the selection of suitable reference genes is imperative to ensure precise gene expression quantification across diverse conditions. This study aims to identify stable reference genes in quinoa seeds under ABA and GA, in order to provide a basis for subsequent research on PHS. Seeds were subjected to different concentrations of ABA and GA (10 μM, 50 μM, 100 μM, and 200 μM). The most suitable treatment concentration was determined based on seed viability. Here, MON1, GAPDH, EIF3, EF1α, ACT, TUB1, and TUB6 were selected as candidate genes. The suitability of these reference genes under different conditions was assessed using various methods including Ct values, geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder. Based on the results obtained from the hormone experiments, it was observed that the application of 100 μM ABA and 200 μM GA yielded the most advantageous outcomes. Additionally, the most appropriate reference genes for different treatments are ACT and TUB1 (H2O treatment), EIF3 and MON1 (ABA, GA treatment and also for the combined data set of the three groups). However, GAPDH exhibited the least stability across all treatments. In summary, ACT is recommended as the reference gene for natural quinoa germination, while EIF3 and MON1 should be used for ABA and GA treatments.

Selection of internal reference gene for quantitative real‐time PCR analysis of gene expression in largemouth bass ( Micropterus salmoides ) under different experimental conditions

The study of gene function in largemouth bass requires accurate normalization by use of suitable internal reference genes. The expression levels of four candidate internal reference genes including β-actin, EF1-α, cytb and 12SrRNA in different tissues, two different developmental stages, and different experimental conditions of largemouth bass were evaluated by quantitative real-time PCR (qPCR) method. The expression stabilities of four candidate internal reference genes were analysed by geNorm, NormFinder and BestKeeper software calculation. The results showed that cytb is the best internal reference gene in different tissues and two different developmental stages of largemouth bass. The cytb and EF1-α was the best choice for internal reference gene in gill of largemouth bass 10 g and 55 g weighing under different salt concentrations respectively. After being challenged by Lipopolysaccharide (LPS), Aeromonas hydrophila and Edwardsiella tarda in largemouth bass weighing 55 g, EF1-α was recommended as the most suitable internal reference gene in spleen, but cytb was ranked as the best internal reference gene in head kidney. The expression levels of CaM normalized by EF1-α and cytb in spleen and head kidney of largemouth bass weighing 55 g, and which showed the significant upregulation in LPS group than those in the control group. Therefore, these results indicated that cytb and EF1-α are the suitable internal reference genes in different tissues, two different developmental stages and different experimental conditions respectively. This study will provide convincing information for the selection of suitable internal reference genes in different tissues, different developmental stages and different experimental conditions of largemouth bass.

Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.

Identification of optimal reference genes in Bombyx mori (Lepidoptera) for normalization of stress-responsive genes after challenge with pesticides.

Pesticides are frequently used to control pests in agriculture due to their ease of use and effectiveness, but their use causes serious economic losses to sericulture when their production overlaps with agriculture. However, no suitable internal reference genes (RGs) have been reported in the study of silkworms in response to pesticides. In this study, a standard curve was established to detect the expression levels of seven RGs in different tissues of different silkworm strains after feeding with pesticides using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), including BmGAPDH, BmActin3, BmTBP, BmRPL3, Bm28sRNA, Bmα-tubulin, and BmUBC, and the stability of them was evaluated by using NormFinder, geNorm, Delta CT, BestKeeper, and RefFinder. The results showed that BmGAPDH and Bmα-tubulin were relatively stable in the midgut after feeding with fenvalerate, BmGAPDH and Bmactin3 were relatively stable in the fat body, and Bmα-tubulin and Bmactin3 were relatively stable in the hemolymph, indicating that Bmactin3 was the most suitable RG when evaluating fenvalerate, followed by BmGAPDH and Bmα-tubulin. Besides, BmGAPDH and Bmactin3 were relatively stable in the midgut after treatment with DDVP, BmGAPDH and Bmα-tubulin were relatively stable in the fat body, and BmGAPDH and Bmα-tubulin were relatively stable in the hemolymph, indicating that Bmα-tubulin was the most stable RG when evaluating DDVP, followed by BmGAPDH and Bmactin3. Of note, BmGAPDH was shared by the two pesticides. The results will be valuable for RG selection in studying the pesticide response mechanism of silkworms and other lepidopteran insects.

Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus.

Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression.

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Selecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

Selection and validation of reference genes for quantitative Real-time PCR analysis in Amana edulis

Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.

Selection and evaluation of reference genes for qRT-PCR of Scutellaria baicalensis Georgi under different experimental conditions

Scutellaria baicalensis Georgi is a famous medicinal plant with its dried roots having been used as a traditional Chinese medicinal for more than 2000 years. Although its genome sequence has previously been published and molecular biology methods have been used to study this species, no suitable internal reference genes have been investigated for standardization of gene expression via quantitative real-time polymerase chain reaction (qRT-PCR). Here, the stabilities of 10 candidate reference genes, ACT11, ACT7, α-TUB, β-TUB, GAPDH, UBC, RPL, SAM, HSP70, and PP2A, were analyzed by four different procedures of GeNorm, NormFinder, BestKeeper, and RefFinder. Their expression stabilities were evaluated under various conditions, including different tissue types (root, stem, leaf, and flower), hormone stimuli treatments (methyl jasmonate, salicylic acid, and abscisic acid), and abiotic stresses (heavy metal, salt, drought, cold, and wounding). The results indicated that β-TUB was the most stable gene for all tested samples, while ACT11 was the most unstable. The most stable reference gene was not consistent under different conditions. β-TUB exhibited the highest stability for different tissue types and abiotic stresses, while for hormone stimuli treatments, ACT7 showed the highest stability. To confirm the applicability of suitable reference genes, we selected to SbF6H and SbF8H as target genes to analyze their expression levels in different tissues. This study helps to the accurate quantification of the relative expression levels of interest genes in S. baicalensis via qRT-PCR analysis.Supplementary InformationThe online version of this article (10.1007/s11033-021-06153-y) contains supplementary material, which is available to authorized users.

ERgene: Python library for screening endogenous reference genes

In gene expression analysis, sample differences and experimental operation differences are common, but sometimes, these differences will cause serious errors to the results or even make the results meaningless. Finding suitable internal reference genes efficiently to eliminate errors is a challenge. Aside from the need for high efficiency, there is no package for screening endogenous genes available in Python. Here, we introduce ERgene, a Python library for screening endogenous reference genes. It has extremely high computational efficiency and simple operation steps. The principle is based on the inverse process of the internal reference method, and the robust matrix block operation makes the selection of internal reference genes faster than any other method.

Internal Reference Gene Selection under Different Hormone Stresses in Multipurpose Timber Yielding Tree Neolamarckia cadamba

Neolamarckia cadamba, a member of the Rubiaceae family, is widely distributed throughout South Asia and South China. In order to acquire reliable and repeatable results, the use of a suitable internal reference gene to normalize the RT-qPCR data is essential. In this study, we reported the validation of housekeeping genes to identify the most suitable internal reference gene(s) for normalization of qPCR data obtained among different tissues (bud, leaf, cambium region) under different hormone stresses. Here, ΔCt, geNorm, NormFinder, and BestKeeper analyses were carried out to analyze the normalization of qPCR data of twenty-one reference gene families (ACT, CAC, CYP, EF1α, eIF, FPS1, FBK, GAPDH, RAN, PEPKR1, PP2A, RPL, RPS, RuBP, SAMDC, TEF, Tub-α, Tub-β, UBCE, UBQ, UPL) including 43 genes. The results showed that FPS1, RPL, and FBK were the most stable reference genes across all of the tested samples. In addition, the expression of NcEXPA8, one gene of interest that plays an important role in regulating cell wall extension, under different phytohormone stresses was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene selection under different phytohormone stresses and a foundation for more accurate and widespread use of RT-qPCR in N. cadamba.

Selection and Validation of Reference Genes for Gene Expression Studies in Codonopsis pilosula Based on Transcriptome Sequence Data

Relative gene expression analyses by RT-qPCR (reverse transcription-quantitative PCR) are highly dependent on the reference genes in normalizing the expression data of target genes. Therefore, inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, and the selection and validation of suitable internal reference genes becomes essential. In this study, we retrieved the commonly used reference genes in transcriptome datasets of Codonopsis pilosula by RNA-Seq (unpublished data), and selected 15 candidate reference genes according to the coefficient of variation (CV) and fold change (FC) value of gene expression. The expression levels of candidate reference genes, which is at different growth stages, undergoing cold stress and drought stress, was determined by RT-qPCR. The expression stability of these genes was evaluated using software packages and algorithms including ΔCt, geNorm, NormFinder and Bestkeeper. Then appropriate reference genes were screened and validated by target gene-UDGPase (UDP glucose pyrophosphorylase). The optimal RGs combinations of C. pilosula, including PP2A59γ, CPY20-1, UBCE32, RPL5B and UBC18 for developmental stage, RPL5B, RPL13 and PP2A59γ for cold treatment, RPL13 and PP2A59γ for drought treatment, were found and proposed as reference genes for future work. This paper laid foundations for both the selection of reference genes and exploration in metabolic mechanism of C. pilosula.

Suitable Reference Genes/miRNAs for qRT-PCR Normalization of Expression Analysis in Sugarcane Under Sorghum mosaic virus Infection

Sugarcane mosaic disease poses a serious threat to the sugarcane industry. Many studies have aimed to unravel the molecular mechanism related to sugarcane and mosaic virus interaction. Quantitative reverse transcription (qRT)-PCR in combination with suitable internal reference genes has been widely used for gene expression analysis. In this study, the expression of 33 candidate reference genes and 12 candidate reference miRNAs was analyzed for first time in the leaf samples of three sugarcane genotype infected with Sorghum mosaic virus using the geNorm, NormFinder and deltaCt (deltaCq) algorithms. A comparison of the expression of eIF-4E and three virus-derived siRNA (vsiR9230S, vsiR9058A and miR16) with the normalized unstable and stable reference genes or miRNA indicated that PP2A and miR159 constituted the best single reference gene/miRNA under SrMV infection. We also suggested that both CUL + CAC and miR171+ miR1520 could serve as the most suitable reference gene/miRNA combination. The use of reliable reference genes and miRNAs should improve the accuracy of gene expression analysis in sugarcane leaves under SrMV stress.

Selection and evaluation of reference genes for gene expression using quantitative real‐time PCR in Mythimna separata walker (Lepidoptera: Noctuidae)

AbstractIn order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real‐time reverse transcription polymerase chain reaction (RT‐qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including β‐actin (β‐ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde‐3‐phosphate (GAPDH), elongation fator‐alpha (EF1α), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha‐tubulin (α‐TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software‐based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web‐based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both RPL7/EF1α were suitable for experiments of different tissues, whereas for insecticide treatments, 28S/α‐TUB were suitable for normalizations of expression data. In addition, 28S/α‐TUB were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

Selection and evaluation of potential reference genes for gene expression analysis in greenbug (Schizaphis graminum Rondani)

In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For greenbug, Schizaphis graminum, a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored. In our study, eight reference genes, elongation fator 1 beta (Ef1β), TATA box binding protein (TBP), alpha-tubulin (α-TUB), 18S ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), actin (ACT), and ribosomal protein L18 (RPL18) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments. To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ΔCt method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene (HSP70), cytocrome P450 gene (SgraCYP18A1), and glutathione S-transferase (GST). We found that the most suitable reference genes varied considerably under different experimental conditions. For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data. In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments. This should be useful for the selection of the suitable reference genes to obtain reliable RT-qPCR data in the gene expression of S. graminum.