Quantification Of Target Gene Expression Research Articles

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

CRISPR deletion of a SINE-VNTR-Alu (SVA_67) retrotransposon demonstrates its ability to differentially modulate gene expression at the MAPT locus.

SINE-VNTR-Alu (SVA) retrotransposons are hominid-specific elements which have been shown to play important roles in processes such as chromatin structure remodelling and regulation of gene expression demonstrating that these repetitive elements exert regulatory functions. We have previously shown that the presence or absence of a specific SVA element, termed SVA_67, was associated with differential expression of several genes at the MAPT locus, a locus associated with Parkinson's Disease (PD) and frontotemporal dementia. However, we were not able to demonstrate that causation of differential gene expression was directed by the SVA due to lack of functional validation. We performed CRISPR to delete SVA_67 in the HEK293 cell line. Quantification of target gene expression was performed using qPCR to assess the effects on expression in response to the deletion of SVA_67. Differences between CRISPR edit and control cell lines were analysed using two-tailed t-test with a minimum 95% confidence interval to determine statistical significance. In this study, we provide data highlighting the SVA-specific effect on differential gene expression. We demonstrate that the hemizygous deletion of the endogenous SVA_67 in CRISPR edited cell lines was associated with differential expression of several genes at the MAPT locus associated with neurodegenerative diseases including KANSL1, MAPT and LRRC37A. This data is consistent with our previous bioinformatic work of differential gene expression analysis using transcriptomic data from the Parkinson's Progression Markers Initiative (PPMI) cohort. As SVAs have regulatory influences on gene expression, and insertion polymorphisms contribute to interpersonal differences in expression patterns, these results highlight the potential contribution of these elements to complex diseases with potentially many genetic components, such as PD.

Induction of a local muscular dystrophy using electroporation in vivo: an easy tool for screening therapeutics

Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose–response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.

Stable reference genes for RT-qPCR analysis of gene expression in the Musa acuminata-Pseudocercospora musae interaction

Leaf pathogens are limiting factors in banana (Musa spp.) production, with Pseudocercospora spp. responsible for the important Sigatoka disease complex. In order to investigate cellular processes and genes involved in host defence responses, quantitative real-time PCR (RT-qPCR) is an analytical technique for gene expression quantification. Reliable RT-qPCR data, however, requires that reference genes for normalization of mRNA levels in samples are validated under the conditions employed for expression analysis of target genes. We evaluated the stability of potential reference genes ACT1, α-TUB, UBQ1, UBQ2, GAPDH, EF1α, APT and RAN. Total RNA was extracted from leaf tissues of Musa acuminata genotypes Calcutta 4 (resistant) and Cavendish Grande Naine (susceptible), both subjected to P. musae infection. Expression stability was determined with NormFinder, BestKeeper, geNorm and RefFinder algorithms. UBQ2 and RAN were the most stable across all M. acuminata samples, whereas when considering inoculated and non-inoculated leaf samples, APT and UBQ2 were appropriate for normalization in Calcutta 4, with RAN and α-TUB most stable in Cavendish Grande Naine. This first study of reference genes for relative quantification of target gene expression in the M. acuminata-P. musae interaction will enable reliable analysis of gene expression in this pathosystem, benefiting elucidation of disease resistance mechanisms.

Selection and validation of reference genes for normalization of qRT-PCR gene expression in wheat (Triticum durum L.) under drought and salt stresses

Eight candidate housekeeping genes were examined as internal controls for normalizing expression analysis of durum wheat (Triticum durum L.) under drought and salinity stress conditions. Quantitative real-time PCR was used to analyse gene expression of multiple stress levels, plant ages (24 and 50 days old), and plant tissues (leaf and root). The algorithms BestKeeper, NormFinder, GeNorm, the delta Ct method and the RefFinder were applied to determine the stability of candidate genes. Under drought stress, the most stable reference genes were glyceraldehyde-3 phosphate, ubiquitin and β-tubulin2, whereas under salinity stress conditions, eukaryotic elongation factor 1-α, glyceraldehyde-3 phosphate and actin were identified as the most stable reference genes. Validation with stress-responsive genes NAC29 and NAC6 demonstrated that the expression level of target genes could be determined reliably with combinations of up to three of the reference genes. This is the first report on reference genes appropriate for quantification of target gene expression in T. durum under drought and salt stresses. Results of this investigation may be applicable to other Triticum species.

PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

Validation of quantitative real-time PCR reference genes for the determination of seasonal and labor-specific gene expression profiles in the head of Western honey bee, Apis mellifera

Honey bee is not only considered an important pollinator in agriculture, but is also widely used as a model insect in biological sciences, thanks to its highly evolved sociality, specialization of labor division, and flexibility of colony management. For an intensive investigation of the seasonal and labor-dependent expression patterns of its genes, accurate quantification of the target gene transcription level is a fundamental step. To date, quantitative real-time PCR (qRT-PCR) has been widely used for rapid quantification of gene transcripts, with reliable reference gene(s) for normalization. To this end, in an attempt to search for reliable reference genes, the amplification efficiencies of six candidate reference genes (rp49, rpL32, rpS18, tbp, tub, and gapdh) were determined. Subsequently, four genes (rpL32, rpS18, tbp, and gapdh) with PCR efficiencies of 90% to 110% were evaluated for their expression stabilities with three programs (geNorm, NormFinder, and BestKeeper) and used for normalization of seasonal expression patterns of target genes in the forager and nurse heads. Although the three programs revealed slightly different results, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR-based determination of seasonal and labor-specific gene expression profiles. Furthermore, the combined use of these two genes yielded a more accurate normalization, compared with the use of a single gene in the head of honey bee. The validated reference genes can be widely used for quantification of target gene expression in honey bee head although it is still remained to be elucidated the expression levels of the selected reference genes in specific tissues in head.

Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach

BackgroundQuantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. No studies have validated specific reference genes in canine osteosarcoma (OS). Previous gene expression studies involving canine OS have used one or two reference genes to normalize gene expression. This study aimed to validate a panel of reference genes commonly used for normalization of canine OS gene expression data using the geNorm algorithm. qPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines.ResultsTumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively.ConclusionsAppropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.

Effect of high energy intake on carcass composition and hypothalamic gene expression in Bos indicus heifers

The objective of this study was to evaluate the effect of high or low energy intake on carcass composition and expression of hypothalamic genes related to the onset of puberty. Twenty-four prepubertal Nellore heifers, 18-20- months-old, with 275.3±18.0 kg body weight (BW), and 4.9±0.2 (1-9 scale) body condition score (BCS) were randomly assigned to two treatments: high-energy diet (HE) and low-energy diet (LE). Heifers were housed in two collective pens and fed diets formulated to promote average daily gain of 0.4 (LE) or 1.2 kg (HE) BW/day. Eight heifers from each treatment were slaughtered after the first corpus luteum detection - considered as age of puberty. The 9-10-11th rib section was taken and prepared for carcass composition analyses. Samples from hypothalamus were collected, frozen in liquid nitrogen, and stored at −80 °C. Specific primers for targets (NPY, NPY1R, NPY4R, SOCS3, OXT, ARRB1, and IGFPB2) and control (RPL19 and RN18S1) genes were designed for real-time PCR and then the relative quantification of target gene expression was performed. High-energy diets increased body condition score, cold carcass weight, and Longissimus lumborum muscle area and decreased age at slaughter. High-energy diets decreased the expression of NPY1R and ARRB1 at 4.4-fold and 1.5-fold, respectively. In conclusion, the hastening of puberty with high energy intake is related with greater body fatness and lesser hypothalamic expression of NPY1 receptor and of β-arrestin1, suggesting a less sensitive hypothalamus to the negative effects of NPY signaling.

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris.

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu and Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes, and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 samples, including 30 staminate and perfect flower samples across developmental stages and 21 different floral tissue samples from mature flowers. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect floral samples. For floral tissues, HIS3, UFD1, and TMP50 were the most suitable reference genes. Furthermore, two target genes, TruPI, and TruFBP24, involved in floral organ identity were selected to validate the most and least stable reference genes in staminate flowers, perfect flowers, and different floral tissues, indicating that the use of inappropriate reference genes for normalization will lead to the adverse results. The reference genes identified in this study will improve the accuracy of qRT-PCR quantification of target gene expression in andromonoecious T. rupestris flowers, and will facilitate the functional genomics studies on flower development and sex differentiation in the future.

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots

To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.

Gene Expressional Profile of Neonatal Neutrophils in Response to Bacterial Lipopolysaccharide (LPS) and Possible Mechanism of Transcription Factor NR4A2 on the Immune Signal Cascade

Gram-negative bacterial infection is a serious condition in neonates which could lead to septicemic shock, disseminated intravascular coagulation and death. LPS, the cell wall component of Gram-negative bacteria is a potent stimulator of the host immune response system, mediated by binding to toll-like receptor (TLR)-4 and CD14 receptors. Neutrophils are the first line of innate defense which possess antimicrobial and acute inflammatory activities. Neonatal neutrophils, though incompletely characterized, are suggested to be functionally immature and compromised in their bactericidal capacity compared with adults.We performed a comparative genome-wide expression array analysis on purified (>95%) human cord blood (term delivery) neutrophils upon challenge by LPS for 4 hours at 100 ng/mL. With the criteria of 2-fold differences and statistical significance (P <0.01), 1236 transcripts were increased and 1633 transcripts were decreased in LPS-treated neutrophils (n=3). A panel of 20 target genes was selected by their magnitude of regulation, novelty and relevant immune functions and validated by qPCR. Eleven of these genes could be linked upstream or downstream of the novel transcription factor, nuclear receptor subfamily-4, group-A, member-2 (NR4A2) by pathway analysis (Metacore). We thus proposed a regulatory network mediated by binding of LPS to TLR4/CD14 receptors, leading to NR4A2 upregulation, thereby promoting a cascade of downstream effector genes associated with antibacterial and/or inflammatory activities in neutrophils.To validate the upstream signal cascade of LPS-induced regulation of NR4A2, neonatal neutrophils were pretreated with specific TLR-4, p38MAPK or NF-κB inhibitors, and stimulated with LPS, followed by quantification of target gene expression by qPCR (n=6 in each group). Our results showed that LPS-induced NR4A2 expression was significantly decreased (all P <0.05) after pretreatment with TLR-4 antibody (0.47 fold), p38MAPK inhibitor SB203580 (0.27 fold), and NF-κB inhibitors BAY-117082 (0.14 fold) and pyrrolidine dithiocarbamate (0.07 fold). These results suggested that NR4A2 could be upregulated by LPS-receptor-mediated p38MAPK and NF-κB signals.To compare the capacity and developmental regulation of LPS-induced responses of NR4A2-associated signals, purified cord blood neutrophils from preterm (gestational age 33-36 weeks) and term (37-40 weeks) neonates, and peripheral blood from healthy male adult (age 25-30 years old) were evaluated (n=10 in each group). LPS-induced expression of NR4A2 and associated molecular signals (EGR1, PPARG, NF-κB, ATOH8, G0S2, GPR84, IL-8, PTGS2, TNF-α, CAMK2G and GCH1) were measured by qPCR. Our result showed that expressions of these target genes were significantly up- or downregulated in LPS-activated neutrophils of neonates and adults. However, at the basal level, expressions of EGR1, GOS2, IL-8 and PTSG2 were significantly lower (P< 0.05) in preterm or term neonatal neutrophils compare with those in adults, showing that there might be compromised levels in neonates at the unstimulated status. Upon stimulation with LPS, expressions of GOS2 and IL-8 were significantly lower (P< 0.05) in preterm or term neonates than in adult, indicating that there could be developmental regulation of NR4A2 downstream genes in neutrophils secondary to LPS stimulations. Correlation analysis showed that the expressions of EGR1 and PTGS2 were positively correlated with NR4A2 in LPS-induced neutrophils from both term (EGR1: R=0.805, P< 0.05; PTGS2: R=0.912, P< 0.05) and preterm neonates (EGR1: R=0.636, P< 0.05; PTGS2: R=0.830, P< 0.05), but not in adult peripheral neutrophils. These results indicated that there could be developmental differences in the regulation of the NR4A2 immune cascade involving apoptosis (EGR1, GOS2)/inflammatory cytokine (IL-8) and prostaglandin synthesis (PTGS2) between adult and neonatal neutrophils at basal and/or LPS-activation states.In summary, we reported the expressional profile and deregulated target genes in neonatal neutrophils upon stimulation by LPS and identified the downstream NR4A2 immune cascade that could be regulated by LPS, p38MAPK and NF-κB signals, as well as the developmental status. DisclosuresNo relevant conflicts of interest to declare.

Reference gene stability of a synanthropic fly, Chrysomya megacephala.

BackgroundStable reference genes are essential for accurate normalization in gene expression studies with reverse transcription quantitative polymerase chain reaction (qPCR). A synanthropic fly, Chrysomya megacephala, is a well known medical vector and forensic indicator. Unfortunately, previous studies did not look at the stability of reference genes used in C. megacephala.ResultsIn this study, the expression level of Actin, ribosomal protein L8 (Rpl8), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1), α-tubulin (α-TUB), β-tubulin (β-TUB), TATA binding box (TBP), 18S rRNA (18S) and ribosomal protein S7 (Rps7) were evaluated for their stability using online software RefFinder, which combines the normal software of the ΔCt method, BestKeeper, Normfinder, and geNorm. Moreover the number of suitable reference gene pairs was also suggested by Excel-based geNorm. The expression levels of these reference genes were evaluated under different experimental conditions with special perspectives of forensic applications: developmental stages (eggs, first, second and third instar larvae, pupae and adults); food sources of larvae (pork, fish and chicken); feeding larvae with drugs (untreated control, Estazolam and Marvelon); feeding larvae with heavy metals (untreated control, cadmium and zinc); tissues of adults (head, thorax, abdomen, legs and wings). According to RefFinder, EF1 was the most suitable reference gene of developmental stages, food and tissues; 18S and GAPDH were the most suitable reference genes for drugs and heavy metals, respectively, which could be widely used for quantification of target gene expression with qPCR in C. megacephala. Suitable reference gene pairs were also suggested by geNorm.ConclusionThis fundamental but vital work should facilitate the gene studies of related biological processes and deepen the understanding in physiology, toxicology, and especially medical and forensic entomology of C. megacephala.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1175-9) contains supplementary material, which is available to authorized users.

Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

RNA Interference for Antimetastatic Therapy.

The suppression of genes involved in tumor progression, metastasis formation, or therapy resistance by RNA interference is a promising tool to treat cancer disease. Efficient delivery of interfering molecules and their sustained presence in tumor cells are required for therapeutic success. This chapter describes a method of systemic application of shRNA expression plasmid via tail vein injection in xenograft mice, causing the sustained reduction of target gene expression in the primary tumor. By choosing S100A4 as a metastasis driving target gene, this therapeutic approach restricted the formation of distant colorectal cancer metastases after intrasplenic transplantation. In vivo imaging of bioluminescent cancer cells allows the monitoring of tumor growth and metastasis formation over time. End point analysis of the trial included scoring of the metastatic burden and the quantification of target gene expression in the tumor. Average S100A4 expression in tumor tissues was reduced by 30 %, causing a 70 % decrease of liver metastases.

Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass (Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ABC], actin [ACTIN], cyclophilin [CYP2], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], beta-amylase 4 [BAM4], zeitlupe [ZTL], MAP Kinase 4 [MPK4], ubiquitin-conjugating enzyme [UBC], S-adenosylmethionine decarboxylase [SAMDC], and translationally controlled tumor protein [TCTP]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN, CYP2, and ABC were found to be the most stably expressed genes for drought stress while ACTIN, TCTP, and ABC were the most stable under salt stress. ACTIN, CYP2, and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2, MPK4, and ABC were most suitable to study waterlogging, and ACTIN, CYP2, and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses.

Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae).

To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

Gene expression profiles in granuloma tissue reveal novel diagnostic markers in sarcoidosis

Sarcoidosis is an immune-mediated multisystem disease characterized by the formation of non-caseating granulomas. The pathogenesis of sarcoidosis is unclear, with proposed infectious or environmental antigens triggering an aberrant immune response in susceptible hosts. Multiple pro-inflammatory signaling pathways have been implicated in mediating macrophage activation and granuloma formation in sarcoidosis, including IFN-γ/STAT-1, IL-6/STAT-3, and NF-κB. It is difficult to distinguish sarcoidosis from other granulomatous diseases or assess disease severity and treatment response with histopathology alone. Therefore, development of improved diagnostic tools is imperative. Herein, we describe an efficient and reliable technique to classify granulomatous disease through selected gene expression and identify novel genes and cytokine pathways contributing to the pathogenesis of sarcoidosis. We quantified the expression of twenty selected mRNAs extracted from formalin-fixed paraffin embedded (FFPE) tissue (n=38) of normal lung, suture granulomas, sarcoid granulomas, and fungal granulomas. Utilizing quantitative real-time RT-PCR we analyzed the expression of several genes, including IL-6, COX-2, MCP-1, IFN-γ, T-bet, IRF-1, Nox2, IL-33, and eotaxin-1 and revealed differential regulation between suture, sarcoidosis, and fungal granulomas. This is the first study demonstrating that quantification of target gene expression in FFPE tissue biopsies is a potentially effective diagnostic and research tool in sarcoidosis.

Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.

Effect of growth hormones on some antioxidant parameters and gene expression in tomato

Bioregulators have a great effect on vital processes of plant growth and development. Known plant bioregulators include Naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA). Natural or synthetic plant bioregulators are organic compounds that affect the physiological processes in the plant, either to control some of these processes or to modify them. For example these bioregulators can affect the nature of the process, either by accelerating or decelerating plant growth, rates of maturation and also by altering the behavior of the plants or their products. Also, enhancement of important nutrients in human diet could be achieved by bioregulators. This study uses the model crop plant Tomato (Lycopersicon esculentum). Tomato is affected by a group of bioregulators, this group contains compounds which are powerful antioxidants in vitro. The current study aims to find out the effect of some plant bioregulators (IAA, IBA and NAA) on tomato growth, total protein content and enzyme activities of ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT). This study also investigates the effect of the above mentioned bioregulators on the level of RNA expression for SOD, CAT and TPX1 genes. The analytical quantification of target gene expression showed the induced effect of NAA on SOD expression and reducing effect of the other bioregulators (IAA and IBA) on CAT and TPX1 expression. However, at the protein level, we found that IBA and IAA caused a minor effect on total protein content while a significant effect was recorded on the total protein level using NAA. Upon measuring the enzyme activity of ascorbate peroxidase and catalase, we found that both the exogenous NAA and IBA stimulated ascorbate peroxidase activity in tomato while there was no considerable difference detected in IAA treated plants. Also, there was no considerable difference detected in catalase activity of all bioregulator-treated plants compared with the control.