Reference Genes For Accurate Normalization Research Articles

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.

Evaluation of reference genes for accurate normalization of qPCR data under biotic stresses in mulberry (Morus indica L.)

Biotic stresses adversely affect the leaf quality as well as the yield of mulberry (Morus spp.) which is used to feed mulberry silkworm (Bombyx mori L.). The different biotic stresses which are of economic importance in mulberry are tukra caused by mealybug insect, root knot disease by nematode, powdery mildew, and root rot by fungi. Reverse transcription-quantitative PCR (RT-qPCR) is a preferred technique to assess the transcript abundance of target genes associated with the diseases using a suitable reference gene as a normalizer. However, no study has been conducted to identify suitable reference genes during different biotic stresses in mulberry. In the present work, six candidate reference genes were quantified through qPCR during different biotic stresses in mulberry and their stability was compared using geNorm, NormFinder and RefFinder algorithms. The analysis of data revealed that actin was the preferred gene during root rot and root knot nematode infection for normalizing gene expression. Ubiquitin gene was the best-scoring gene for mealybug infestation resulting in tukra disease. PP2A (Protein phosphatase 2A) and RPL3 (60S ribosomal protein L3) were highly stable expressing genes during powdery mildew fungi infection. In the combined analysis of all tested biotic stresses, actin gene was the most preferred one followed by ubiquitin and PP2A as reference genes. This study identified the best scoring reference genes in various biotic stresses which will contribute for the precise quantification of target genes by RT-qPCR in mulberry as a tool to unravel the disease resistance mechanism.

The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus.

Quantitative real-time PCR (qPCR) is a sensitive and commonly used technique for gene expression profiling and provides insight into biological systems. Successful qPCR requires the use of appropriate reference genes for the normalization of data. In the present study, we aimed to identify and assess the best-suited reference genes in near-isogenic resistant (R) and susceptible (S) tomato lines infected with begomovirus Tomato curly stunt virus (ToCSV). Ten candidate reference genes namely, Actin7 (ACT), β-6 Tubulin (TUB), Ubiquitin 3 (UBI), Clathrin adaptor complexes medium subunit (CAC), Phytoene desaturase (PDS), Expressed protein (EXP), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Adenine phosphoribosyl transferase-like protein (APT1), TAP42-interacting protein (TIP41) and Elongation factor 1-alpha (EF1α) were selected and evaluated for their expression stability in resistant and susceptible tomato leaves using the analytical tools geNorm, NormFinder, BestKeeper, and RefFinder. After ranking the reference genes from most to least stable, the results suggested that a combination of ACT, EXP, and EF1α in the S lines and a combination of TIP41, APT1, and ACT in the R line is appropriate for qPCR normalization. Furthermore, to validate the identified reference genes, iron superoxide dismutase (SOD), heat shock protein 70 (HSP70) and Glutathione-S-transferase (GST) were selected as targets for normalization. The relative expression of the target genes varied when normalized against the most stable reference genes in comparison to the least stable genes. These results highlight the importance of careful selection of reference genes for accurate normalization in qPCR studies.

Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (Eriobotrya japonica)

Loquat (Eriobotrya japonica Lindl.) is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components. Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression. In this study, eight candidate reference genes were selected from our previously published RNA-seq data, and primers for each candidate reference gene were designed and evaluated. The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples, including 12 subgroups of developing or abiotic-stressed tissues. Different combinations of stable reference genes were screened according to a comprehensive rank, which was synthesized from the results of four algorithms, including the geNorm, NormFinder, BestKeeper and ΔCt methods. The screened reference genes were verified by normalizing EjLGA1 in each subgroup. The obtained suitable combinations of reference genes for accurate normalization were GAPDH, EF1α and ACT for floral development; GAPDH, UBCE and ACT for fruit setting; EF1α, GAPDH and eIF2B for fruit ripening; ACT, EF1α and UBCE for leaves under heat stress; eIF2B, UBCE and EF1α for leaves under freezing stress; EF1α, TUA and UBCE for leaves under salt stress; ACT, EF1α and eIF2B for immature pulp under freezing stress; ACT, UBCE and eIF2B for immature seeds under freezing stress; EF1α, eIF2B and UBCE for both immature pulp and seeds under freezing stress; UBCE, TUB and TUA for red-fleshed fruits under cold-storage stress; eIF2B, RPS3 and TUB for white-fleshed fruits under cold-storage stress; and eIF2B, UBCE and RPS3 for both red- and white-fleshed fruits under cold-storage stress. This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat. To our knowledge, this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E. japonica. The use of the three most stable reference genes could increase the reliability of future quantification experiments.

Expression Pattern of Entire Cytochrome P450 Genes and Response of Defenses in a Metabolic-Herbicide-Resistant Biotype of Polypogon fugax.

Enhanced herbicide metabolism mediated by cytochrome P450s has been proposed as one of the major mechanisms of resistance to fenoxaprop-P-ethyl in a metabolic-herbicide-resistant biotype of Asia minor bluegrass (Polypogon fugax Nees ex Steud.). Upon pre-treatment with the P450 inhibitor piperonyl butoxide, a remarkable reduction in metabolic rates of the phytotoxic fenoxaprop-P has been observed in the resistant plants, implying that constitutive and/or fenoxaprop-P-ethyl-induced up-regulation of specific P450 isoforms are involved in the fenoxaprop-P-ethyl resistance. However, which P450 gene(s) were responsible for the metabolic resistance is still unknown. In this present study, based on the abundant gene resources of P. fugax established previously, a total of 48 putative P450 genes were isolated from the metabolic-herbicide-resistant plants and used for gene expression analysis. The most suitable reference genes for accurate normalization of real-time quantitative PCR data were first identified in P. fugax and recognized as actin (ACT), 18S rRNA (18S), and ribulose-1,5-bisphosphate carboxylase oxygenase (RUBP) under fenoxaprop-P-ethyl stress, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1α (EF1α) under mesosulfuron-methyl stress, and ACT, EF1α, eukaryotic initiation factor 4a (EIF4A), and 25S rRNA (25S) at different growth stages. Expression analysis of the putative P450 genes revealed that six genes, respectively, annotated as CYP709B1, CYP71A1-4, CYP711A1, CYP78A9, P450-11, and P450-39 were up-regulated more than 10-fold in the resistant plants by fenoxaprop-P-ethyl treatment, and all of them exhibited constitutively and/or herbicide-induced higher transcript levels in the fenoxaprop-P-ethyl-resistant than in the susceptible plants. Three genes, respectively, annotated as CYPRO4, CYP313A4, and CYP51H11 constantly up-regulated in the resistant than in the susceptible plants after fenoxaprop-P-ethyl treatment. Up-regulated expressions of these specific P450 genes were consistent with the higher P450 contents determined in the resistant plants. These results will help to elucidate the mechanisms for P450-mediated metabolic-herbicide resistance in P. fugax as well as other grass weed species.

A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma

Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.

Validation of common reference genes stability in exosomal mRNA-isolated from liver and breast cancer cell lines.

Accurate relative gene expression analysis by reverse transcription-quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well-known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes-isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK-BR-3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes-isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium

Accurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

Identification of reference genes for real-time polymerase chain reaction gene expression studies in Nile rats fed Water-Soluble Palm Fruit Extract.

The Nile rat (Arvicanthis niloticus) is a novel diurnal carbohydrate-sensitive rodent useful for studies on type 2 diabetes mellitus (T2DM) and the metabolic syndrome. Hepatic responses to T2DM and any interventions thereof can be evaluated via transcriptomic gene expression analysis. However, the study of gene expression via real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) requires identification of stably expressed reference genes for accurate normalisation. This study describes the evaluation and identification of stable reference genes in the livers from Control Nile rats as well as those supplemented with Water-Soluble Palm Fruit Extract, which has been previously shown to attenuate T2DM in this animal model. Seven genes identified as having stable expression in RNA-Sequencing transcriptome analysis were chosen for verification using real-time RT-qPCR. Six commonly used reference genes from previous literature and two genes from a previous microarray gene expression study in Nile rats were also evaluated. The expression data of these 15 candidate reference genes were analysed using the RefFinder software which incorporated analyses performed by various algorithms. The Hpd, Pnpla6 and Vpp2 genes were identified as the most stable across the 36 samples tested. Their applicability was demonstrated through the normalisation of the gene expression profiles of two target genes, Cela1 and Lepr.In conclusion, three novel reference genes which can be used for robust normalisation of real-time RT-qPCR data were identified, thereby facilitating future hepatic gene expression studies in the Nile rat.

Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures.

Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20–22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats, Pgk1, Ppia, and B2m expression are the most stable within the medial prefrontal cortex; Ppia, Rpl13a, and Sdha within the temporal cortex; Pgk1, Ppia, and Rpl13a within the entorhinal cortex; Gapdh, Ppia, and Pgk1 within the dorsal hippocampus; Rpl13a, Sdha, and Ppia within the ventral hippocampus; and Sdha, Pgk1, and Ppia within the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.

Evaluation of internal reference genes in Auxenochlorella protothecoides under continuous heterotrophic culture conditions at normal, low and high temperatures

Real-time reverse quantitative transcription PCR (RT-qPCR) is a commonly used method for gene expression analysis. However, the accuracy of RT-qPCR analysis greatly depends on the stability of the reference genes selected for data normalization. Unfortunately, there have been no systematic evaluation studies on reference genes in the unicellular green alga Auxenochlorella protothecoides. Ten genes (ACT, COX6B, CYB5R4, EF1α, GAPDH, rbcL, RPL19, RPL32, RPS10 and TUA) were chosen as candidate reference genes. The expression stability of each reference gene was determined by applying geNorm, NormFinder, BestKeeper and comprehensive ranking based on geomean ranking values. In order to validate the suitability and reliability of the candidate reference genes selected for data normalization, two key functional genes related to lipid metabolism were also selected as target genes. The results revealed that RPL32 was the most stably expressed and suitable reference genes under all experimental conditions of the analyses; CYB5R4 was also a suitable reference gene under continuous heterotrophic culture condition, and RPL19 was top-ranked and stably expressed under both low and high temperature stress. These results provided a list of suitable reference genes for accurate normalization of heterotrophic A. protothecoides time-course samples under different temperature stress in qPCR experiments. This study will be of very great reference value to assess the stability of candidate reference genes in other heterotrophically grown algae.

Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples

RT-qPCR requires an adequate choice of stably expressed reference genes for accurate normalization of mRNA expression. However, testing a panel of reference genes is often time-consuming and expensive. In this work, we aimed to develop a set of multiplex real-time PCR assays for RT-qPCR analysis of commonly used housekeeping genes in laboratory rats. Using Hydrolysis probe (TaqMan®) technology, we have designed and optimized three triplex qPCR assays (Actb + Gapdh + B2m; Rpl13a + Sdha + Ppia; Hprt1+Pgk1+Ywhaz) demonstrating optimal PCR amplification efficiencies (from 94.7 to 100.5%) and repeatability. Novel assays allow expression analysis of 9 reference genes in 3 reactions making possible a more time-efficient choice of reference genes in RT-qPCR experiments in Wistar rats in comparison with widespread singleplex assays.

Evaluation of global expression of selected genes as potential candidates for internal normalizing control during transcriptome analysis in dromedary camel (camelus dromedarius)

Abstract Background Selection of the stable normalizing genes is critical for quantification of gene expression. Unfortunately, till date, no report on systematic assessment of reference genes is known for dromedary camel (Camelus dromedaries). Therefore, this is the first study to validate the stability of five potential reference genes in different tissues of Camel. Results The expression levels of the glyceraldehyde-3P-dehydrogenase (GAPDH), TUBB (β-tubulin), H-Ras protein, c-Myc and ACTB (β-actin) transcripts were quantified in the liver, brain, heart, spleen, kidney, adipose tissue, and skeletal muscle. Based on the stability ranking, performed with the geNorm VBA applet for Microsoft Excel, Normfinder and ΔCt method, the examined genes were arranged as follows GAPDH > ACTB > TUBB >H-Ras > c-Myc. Conclusion Our findings suggest that the GAPDH and ACTB genes can be used as stable reference genes for accurate normalization of gene expression data using qRT-PCR in the Camelus dromedarius.

Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

Reference genes for accurate normalization of gene expression in wood-decomposing fungi

Wood-decomposing fungi efficiently decompose plant lignocellulose, and there is increasing interest in characterizing and perhaps harnessing the fungal gene regulation strategies that enable wood decomposition. Proper interpretation of these fungal mechanisms relies on accurate quantification of gene expression, demanding reliable internal control genes (ICGs) as references. Commonly used ICGs such as actin, however, fluctuate among wood-decomposing fungi under defined conditions. In this study, by mining RNA-seq data in silico and validating ICGs in vitro using qRT-PCR, we targeted more reliable ICGs for studying transcriptional responses in wood-decomposing fungi, particularly responses to changing environments (e.g., carbon sources, decomposition stages) in various culture conditions. Using the model brown rot fungus Postia placenta in a first-pass study, our mining efforts yielded 15 constitutively-expressed genes robust in variable carbon sources (e.g., no carbon, glucose, cellobiose, aspen) and cultivation stages (e.g., 15 h, 72 h) in submerged cultures. Of these, we found 7 genes as most suitable ICGs. Expression stabilities of these newly selected ICGs were better than commonly used ICGs, analyzed by NormFinder algorithm and qRT-PCR. In a second-pass, multi-species study in solid wood, our RNA-seq mining efforts revealed hundreds of highly constitutively expressed genes among four wood-decomposing fungi with varying nutritional modes (brown rot, white rot), including a shared core set of ICGs numbering 11 genes. Together, the newly selected ICGs highlighted here will increase reliability when studying gene regulatory mechanisms of wood-decomposing fungi.

Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars

ABSTRACT: The aim of this study was to analyze the expression of putative reference genes in sugarcane under drought stress. The varieties RB72454 and RB72910 were cultivated and the treatments control and drought stress applied to 135-day-old plants grown under field conditions. The stress level of the plants was measured by rate of photosynthesis, transpiration, and stomatal conductance. For each biological replicate, expression analyses were conducted using quantitative polymerase chain reaction for the genes α-tubulin, β-tubulin, β-actin, cyclophilin, eukaryotic elongation factor 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 and ubiquitin. Stability was evaluated using the geNorm, NormFinder, and BestKeeper software packages. Among the candidate genes, GAPDH was identified as the most stable in all software, indicating its suitability for gene expression studies in sugarcane undergoing drought stress; the gene β-actin was the second most stable. These findings suggest using GAPDH and β-actin for normalization in relative gene expression in sugarcane.

Validation of reference genes for accurate normalization of gene expression with quantitative real-time PCR in Haloxylon ammodendron under different abiotic stresses.

Haloxylon ammodendron plays an important role in maintaining the structure and function of the entire ecosystem where it grows. No suitable reference genes have been reported in H. ammodendron plants to date. In this study, a total of 8 reference genes (18S, ACT1, ACT7, UBC18, TUA5, GAPDH, EF-1α and UBQ10) were selected from the available trancriptome database, and the expression stability of these 8 candidate genes was validated under different abiotic stress with three different statistical algorithms (geNorm, NormFinder and BestKeeper). The results produced from different models were in agreement with each other essentially: 18S and TUA5 were the most stable genes under drought stress, 18S, the most stable gene under heat stress and mechanical damage, ACT7 and UBC18, stable under salt stress while TUA5 and GAPDH expressed constantly under mechanical damage, and ACT1 expressed steadily under cold conditions. Expression profiles of several stress response genes, including FT-5, FT-9, DREB2A and DREB2C, were further confirmed with various candidate reference genes. None of the candidate genes showed a constant expression among all tested samples. Hence, it's essential to use more than one reference gene in order to guarantee the accuracy of quantitative real-time PCR. The results of this study will contribute to the accuracy and reliability in transcripts quantification, which is of significance to transcription-based studies and applications in this important shrub H. ammodendron.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis–an obligate aphid pathogenic fungus—the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Validation of Reliable Reference Genes for Accurate Normalization in RT-qPCR Analysis of Codonopsis pilosula

ObjectiveTo identify reliable reference genes (RGs) for normalization of real-time PCR (RT-qPCR) data in Codonopsis pilosula. MethodsThe expression profiles of 10 candidate RGs (GAPDH, ACT, α-TUB, β-TUB, UBQ, CYP, EF-1α, NAC, F-box and PP2A) were examined in C. pilosula during the phenological period. The raw materials examined included roots, stems, leaves, and flower buds at flowering and boll-forming stages, five growth stages of untreated and treated roots with plant growth retardant. The best-suited RGs were accessed using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. ResultsThe best-ranked references genes differed across the samples. GAPDH and PP2A were the most suitable for expression analysis in untreated tissues while GAPDH, α-TUB, and PP2A were ranked as the three most stably expressed genes in untreated roots, while NAC and CYP were the most stably expressed genes in stressed (i.e., treated) roots. The expression of UGPase, a key enzyme for CPP biosynthesis, was determined to further validate the selected RGs. ConclusionA total of 10 RGs can be used as reference genes of C. pilosula, however the appropriate one should be used as it may chance.