Relative Quantification Of Gene Expression Research Articles

Excreted and secreted products (72/60 kDa) from Haemonchus placei larvae induce in vitro peripheral blood mononuclear cell proliferation and activate the expression of cytokines and FCεR1A receptor

The aim of the present study was to assess the expression of cytokines and FCεR1A receptor stimulated by Haemonchus placei larval excretory and secretory (ES) products associated with the pathogenesis in calves. Bovine peripheral blood mononuclear cells (PBMC) were stimulated in in vitro assays with H. placei L4 ES product at 8, 12, 16 and 24 h. ES products were collected in in vitro assays at 48 h with molecular weight of 72/60 kDa and isoelectric point of 7.2 pI. Specific IgG for infected and control calves, positive and negative, were employed to recognise H. placei larval ES products by indirect ELISA, showing a mean of 1.8, 0.83 and 0.28 OD, respectively, (p ≤ 0.001). The quantification of relative gene expression was performed using a set of cytokines (IL-2, IFNγ, TGFβ, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13), FCεR1A receptor and housekeeping (GAPDH, β-actin and β-2-microglobulin) by RT-qPCR. An early increased expression, 2.2- to 3.4-fold change, of IL-2 (p ≤ 0.001), IL-5 and TGFβ (p ≥ 0.05) was determined, followed by TGFβ (30.7 and 14.14), IL-8 (102.8 and 1504.4) and IL-10 (60.4 and 1.7) (p ≤ 0.05) after 12 and 16 h, respectively, and reducing the expression level at 24 h. In addition, IL-6, IL-13 and FCεR1A receptor also displayed mild expression level, 2.1 – to 7.60-fold change, at 24 h (p ≥ 0.05). We conclude that ES products of 72/60 kDa collected in vitro from H. placei larvae are recognised by infected hosts and have the ability to induce diverse immune factors to modulate the nematode damage.

Gene expression of some cytokines in patients with Cutaneous leishmaniasis in Al-Diwanyah province

The current study was conducted in the Department of Biology - Faculty of Education for Girls - University of Kufa for the period from March 2018 to August 2018, which aims to detect the genetic expression of the mRNA of some of the cytokines in the peripheral blood of patients with cutaneous leishmaniasis using RT-qPCR technique. The results showed that the relative quantification of gene expression of mRNA for IFN-γ, IL-4, IL-10 andTNF-α in the blood of patients with cutaneous leishmaniasis compared to control. The highest amount was TNF-α, followed byIFN-γ, IL-10 and IL-4, which was 15.9±4.9, 10.5±4.6, 7.6±3.7 and 3.7±1.1 respectively, compared with control of 2.3±1.2, 2.6±1.2, 2.6±1.2 and 1.9±1.3 respectively, with a statistically significant difference at P <0.05. The correlation results showed that all cytokines (IFN-γ, IL-4, IL-10, TNF-α) were significantly correlated with each other in terms of gene expression and the association was strong betweenIFN-γ and IL-10 (r = 0.88), followed by IFN-γand TNF-α (r = 0.75) and IL-10 and TNF-α, (r = 0.63) and then IL-4 and IL-10 (r = 0.54).The conclusion from this study that a cellular immune response is achieved by increasing the genetic expression of cytokines, IFN-γ, IL-4, IL-10 and TNF-α in the peripheral blood of patients with cutaneous leishmaniasis and all these cytokines are positively correlated with each other.

A cluster of CYP6 gene family associated with the major quantitative trait locus is responsible for the pyrethroid resistance in Culex pipiens pallen.

The emergence and rapid spread of insecticide resistance in several mosquito species has become a significant obstacle in management of mosquito-borne diseases, including deltamethrin resistance in Culex pipiens pallens. Previous study identified a major deltamethrin resistance quantitative trait locus (DR-6) that alone explained 62% of the genetic variance. In this study, the marker L4B1.102 and L4B1.175 associated with the DR-6 were characterized. We searched for potential candidate genes in the flank region of two markers in the genome sequence and showed that a cluster of CYP6 cytochrome P450 genes (CYP6BB4, CYP6BB3, CYP6CC2, CYP6P14, CYP6BZ2, CYP6AA9, CYP6AA8, CYP6AA7) was in the vicinity of DR-6. Significant differences in the expression of these P450s in the larval and adult stages were identified in the resistant strains compared with the susceptible strain. For CYP6AA9 and CYP6BB4, the correlation analysis showed a highly positive correlation between relative gene expression quantification and the resistance level in different strains. Knockdown of CYP6BB4 increased the sensitivity of mosquitoes to deltamethrin. We identified that the deltamethrin resistance was in a cluster of CYP6 genes in C.pipiens pallens, and CYP6BB4 may play a significant role in the development of deltamethrin resistance.

Gene expression of Interleukin-18 in rheumatoid arthritis patients on disease modifying anti-rheumatic drug therapy.

Background & Objectives:The hallmark of rheumatoid arthritis is the inflammation that is mediated by the macrophages and monocytes that cause release of pro-inflammatory cytokines like interleukin-18. It is highly expressed in serum of patients suffering from rheumatoid arthritis and has a positive association with disease activity. The aim of this study was to analyze the gene expression of interleukin-18 in rheumatoid arthritis patients on disease modifying anti-rheumatic drug therapy.Methods:The cross sectional comparative study is conducted at Department of Biochemistry and Molecular Biology and Center for Research in Experimental and Applied Medicine (CREAM-1Lab), Army Medical College, Rawalpindi, in collaboration with Rheumatology Department, Military Hospital, Rawalpindi. Study was conducted on two groups consisting of Group-I of rheumatoid arthritis patients on diseases modifying anti-rheumatic drugs and control Group-II comprising of normal healthy individuals. Non-probability purposive sampling was done from patients and controls. The duration of study was one year i-e from November 2015 to November 2016. Relative quantification of gene expression of interleukin-18 was done by Real time PCR using ∆∆CT method.Results:Expression analysis for interleukin-18 showed down regulation of gene in rheumatoid arthritis patients as compared to controls.Conclusion:Interleukin-18 gene shows down regulation in rheumatoid arthritis patients on disease modifying anti-rheumatic drugs therapy.

Validation of reference genes for reverse transcription real-time quantitative PCR analysis in the deep-sea bacterium Shewanella psychrophila WP2.

Reference genes are critical to obtain reliable results of reverse transcription real-time quantitative PCR (RT-qPCR), which is widely used for relative quantification of gene expression. In this study, we evaluated the validity of seven candidate reference genes for normalization in RT-qPCR analysis in the deep-sea bacterium Shewanella psychrophila WP2 under different environmental conditions. Among the set of genes investigated, gyrA, 16S rRNA and rho were identified as the most suitable reference genes for WP2 at different temperatures, hydrostatic pressures and salinities, respectively. Notably, the rho gene is conserved in Shewanella genus and other deep-sea bacteria, thus, could be used as a versatile reference gene for RT-qPCR analysis of these microorganisms under extreme environmental conditions.

Expression of prolactin receptors in the duodenum, kidneys and skeletal system during physiological and sulpiride-induced hyperprolactinaemia.

Hyperprolactinaemia in pregnancy leads to mild and reversible changes in the maternal skeletal system, and medicamentous hyperprolactinemia causes more detrimental effects. We conducted an experimental study to evaluate differences between Prlr gene expression in the duodenum, vertebrae and kidneys during physiological and medicamentous hyperprolactinaemia, which could influence calcium homeostasis. Experimental animals (18 weeks old, Wistar female rats) were divided as follows: group P (nine rats that were 3 weeks pregnant), group M (ten rats that were intramuscularly administrated sulpiride (10 mg/kg) twice daily for 3 weeks), and the control group (C, ten age-matched nulliparous rats, 18-week-old). Laboratory investigations included measurements of serum ionized calcium, phosphorus, urinary calcium and phosphorus excretion, osteocalcin (OC), serum procollagen type 1 N-terminal propeptide (P1NP), vitamin D, parathyroid hormone (PTH) and prolactin (PRL). Relative quantification of gene expression for prolactin receptors in the duodenum, vertebrae and kidneys was determined using real-time PCR. Expression of the Prlr gene was significantly higher in the duodenum (p < 0.001) and lower in vertebrae (p < 0.001) and kidneys (p < 0.01) in rats with physiological hyperprolactinaemia (PHP) than in the control group. Significantly lower Prlr expression in the duodenum was verified (p < 0.001), along with increased Prlr gene expression in vertebrae (p < 0.001) and kidneys (p < 0.01), in rats with medicamentous hyperprolactinaemia (MHP) than in the C group. Downregulation of Prlr gene expression in the duodenum may explain the diminished intestinal calcium absorption in medicamentous hyperprolactinaemia. Prolactin takes calcium from the skeletal system following increased Prlr gene expression in the vertebrae to maintain calcium homeostasis, which increases the harmful effect on bone metabolism compared to that of physiological hyperprolactinaemia.

MLL-rearranged acute myeloid leukemia: Influence of the genetic partner in allo-HSCT response and prognostic factor of MLL 3' region mRNA expression.

MLL gene is involved in more than 80 known genetic fusions in acute leukemia. To study the relevance of MLL partner gene and selected gene's expression, in this work, we have studied a cohort of 20 MLL-rearranged acute myeloid leukemia (AML). Twenty MLL-rearranged AML patients along with a control cohort of 138 AML patients are included in this work. By RT-PCR and sequencing, MLL genetic fusion was characterized, and relative gene expression quantification was carried out for EVI1, MEIS1, MLL-3', RUNX1, SETBP1, HOXA5, and FLT3 genes. Risk stratification and association of MLL genetic partner and gene expression to overall survival, in the context of received therapy, were performed. MLLr cohort showed to have an OS more similar to intermediate-risk AML. Type of MLL genetic partner showed to be relevant in allo-HSCT response; having MLLT1 and MLLT3, a better benefit from it. Expression of MLL-3' region, EVI1 and FLT3, showed association with OS in patients undergoing allo-HSCT. We show that the MLL genetic partner could have implications in allo-HSCT response, and we propose three genes whose expression could be useful for the prognosis of this leukemia in patients undergoing allo-HSCT: 3' region of MLL, EVI1, and FLT3.

Validation of reference genes for reverse transcription real-time quantitative PCR analysis in the deep-sea bacterium Shewanella psychrophila WP2

Reference genes are critical to obtain reliable results of reverse transcription real-time quantitative PCR (RT-qPCR), which is widely used for relative quantification of gene expression. In this study, we evaluated the validity of seven candidate reference genes for normalization in RT-qPCR analysis in the deep-sea bacterium Shewanella psychrophila WP2 under different environmental conditions. Among the set of genes investigated, gyrA, 16S rRNA and rho were identified as the most suitable reference genes for WP2 at different temperatures, hydrostatic pressures and salinities, respectively. Notably, the rho gene is conserved in Shewanella genus and other deep-sea bacteria, thus, could be used as a versatile reference gene for RT-qPCR analysis of these microorganisms under extreme environmental conditions.

Effect of Melatonin on the stability and expression of reference genes in Catharanthus roseus

The role of Melatonin in influencing diverse genes in plants has gained momentum in recent years and many reports have employed qRT-PCR for their quantification. Relative quantification of gene expression relies on accurate normalization of qRT-PCR data against a stably-expressing internal reference-gene. Although researchers have been using commonly available reference-genes to assess Melatonin-induced gene expression, but to-date, there have been no attempts to validate the reference-gene stability under Melatonin-supplementation in planta. In this study, we performed stability assessment of common reference-genes under Melatonin-supplementation and abiotic stress in leaves and seedlings of Catharanthus roseus using geNorm, NormFinder, BestKeeper, ΔCt and RefFinder algorithms. Nine candidate reference-genes were tested for stability in C. roseus (FBOX, CACS, TIP, RSP9, EXP, EXPR, SAND, F17M5, ACT) and our study inferred that while EXP and EXPR were the most-stable, F17M5 was the lowest-stable gene in the leaf-fed samples. Among seedlings of C. roseus, F17M5 and TIP were the most, while ACT was the least-stable gene. The suitability of selected stable reference-gene pairs was demonstrated by assessing the transcript levels of the Melatonin-biosynthesis gene SNAT under same conditions. Our study is the first to comprehensively analyze the stability of commonly-used reference-genes under Melatonin-induced conditions in C. roseus.

Zerumbone suppresses the activation of inflammatory mediators in LPS-stimulated U937 macrophages through MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways

Zerumbone (ZER), isolated mainly from the Zingiber zerumbet (Z. zerumbet) rhizomes was found to be effective against numerous inflammatory and immune disorders, however, the molecular and biochemical mechanisms underlying its anti-inflammatory and immunosuppressive properties have not been well studied. This study was carried out to examine the profound effects of ZER on inflammatory mediated MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways in LPS-stimulated U937 human macrophages. ZER significantly suppressed the up-regulation pro-inflammatory mediators, TNF-α, IL-1β, PGE2, and COX-2 protein in LPS-induced human macrophages. Moreover, ZER significantly downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/β as well as restored the degradation of IκBα. ZER correspondingly showed remarkable attenuation of the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a concentration-dependent manner. ZER also diminished the expression of upstream signaling molecules TLR4 and MyD88, which are prerequisite for the NF-κB, MAPK and PI3K-Akt activation. Additionally, quantification of relative gene expression of TNF-α, IL-1β, and COX-2 indicated that, at a higher dose (50μM), ZER significantly downregulated the elevated mRNA transcription levels of the stated pro-inflammatory markers in LPS-stimulated U937 macrophages. The strong suppressive effects of ZER on the activation of inflammatory markers in the macrophages via MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways suggest that ZER can be a preventive and potent therapeutic candidate for the management of various inflammatory-mediated immune disorders.

Longevity-Related Gene Transcriptomic Signature in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) (grade IV astrocytoma) has been assumed to be the most fatal type of glioma with low survival and high recurrence rates, even after prompt surgical removal and aggressive courses of treatment. Transcriptional reprogramming to stem cell-like state could explain some of the deregulated molecular signatures in GBM disease. The present study aimed to quantify the expression profiling of longevity-related transcriptional factors SOX2, OCT3/4, and NANOG to evaluate their diagnostic and performance values in high-grade gliomas. Forty-four specimens were obtained from glioblastoma patients (10 females and 34 males). Quantitative real-time polymerase chain reaction was applied for relative gene expression quantification. In silico network analysis was executed. NANOG and OCT3/4 mRNA expression levels were significantly downregulated while that of SOX2 was upregulated in cancer compared to noncancer tissues. Receiver operating characteristic curve analysis showed high diagnostic performance of NANOG and OCT3/4 than SOX2. However, the aberrant expressions of the genes studied were not associated with the prognostic variables in the current population. In conclusion, the current study highlighted the aberrant expression of certain longevity-associated transcription factors in glioblastoma multiforme which may direct the attention towards new strategies in the treatment of such lethal disease.

Evaluation of the miRNA-146a and miRNA-155 Expression Levels in Patients with Oral Lichen Planus.

Oral Lichen Planus (OLP) is a chronic autoimmune disease that could be considered as a potential premalignant status. To evaluate the miRNA-146a and miRNA-155 expression levels in patients with oral Lichen planus lesions compared to healthy subjects with normal oral mucosa. Forty patients with oral lichen planus and 18 healthy age and gender-matched controls were recruited in this case-control study. Oral lichen planus was diagnosed clinically and pathologically. The expression levels of two miRNAs in peripheral blood samples were determined using commercial TaqMan MicroRNA Assays. Relative quantification of gene expression was calculated by the 2-ΔΔct method. The expression levels of miRNA-146a and miRNA-155 in patients with oral Lichen planus were significantly higher than those of healthy controls. Also, a direct but insignificant correlation was found between miRNA-155 and miRNA-146a expression levels among the patient group. Our findings indicate that miRNA-146a and miRNA-155 could be potential biomarkers for the immunopathogenesis of oral lichen planus.

Abstract 1477: Melatonin regulates the tumor suppressor miR-148a-3p involved in angiogenesis and metastasis of breast cancer

Abstract Women with breast cancer has the tumor progression and angiogenesis-induced metastasis as the main cause of death. MicroRNAs (miRNAs) are small noncoding mRNA molecules that play an important role in gene regulation and once deregulated, these molecules may be involved with the progression of different human tumor types, including breast cancer. These miRNAs play an oncostatic role on tumor suppressor genes and regulate the process of angiogenesis, tumor growth and metastasis. So as new possible adjuvant treatment against breast cancer our group have shown melatonin, that is a hormone secreted by the pineal gland, by exhibiting several anti-tumor and antiangiogenic effects. Therefore, the aim of this study was to evaluate the potential therapeutic of melatonin on miRNAs regulation to verify breast cancer progression and potent tumor suppressor miR-148a-3p. In silico analysis was performed for selection of miRNAs involved in breast cancer. The MDA-MB-231 breast cancer cell line (metastatic negative estrogen receptor) was grown and separated in two different experimental conditions maintained for 24 hours: control group and melatonin-treated group (concentration of 1 mM). After this period the extraction of total RNA was performed (Qiagen®) and the total concentration of miRNAs of each sample was determined (NanoDrop Spectrophotometer 2000C - Thermo Scientific®). Differential expressions of these miRNAs was evaluate using miScript miRNA PCR-Array (Qiagen®) containing 84 miRNAs associated with breast cancer. The overexpression of miR-148a-3p in MDA-MB-231 cells was performed by bacterial cloning vector and the relative quantification of gene expression of their target IGF-1R and VEGF by real-time PCR. Moreover, the quantification of protein expression was performed by immunocytochemistry of IGF-1R and VEGF. Lastly, the migration and invasion cell assays were carried out to assess the potential capacity of these cells to undergo metastasis. The analysis of miRNAs MDA-MB-231 cell line by PCR-array showed 24 upregulated miRNAs and 8 miRNAs downregulated after the treatment with melatonin. Relative quantification shows that the melatonin treatment increases the gene expression of miR-148a-3p and decreases the gene and protein levels of IGF-1R and VEGF. The capability of migration and invasion of the breast cancer cells decreased after treatment with melatonin and overexpression of miR-148a-3p. Our results confirm the action of melatonin on the miR-148a-3p regulation known being involved in the progression of breast cancer. Thus the establishment of this therapeutic protocols can control these cellular events essential for the prognosis of patients with breast cancer. Citation Format: Debora Zuccari, Jéssica Zani Lacerda, Lívia Carvalho Ferreira, Beatriz Camargo Lopes, Andrés Felipe Aristizábal-Pachón, Marcio Chaim Bajgelman. Melatonin regulates the tumor suppressor miR-148a-3p involved in angiogenesis and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2017-1477

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands).

P3.03-039 Prognostic Biomarkers for Malignant Pleural Mesothelioma Treated with Chemotherapy: Topic: Mesothelioma Clinical

Prognosis of Malignant Pleural Mesothelioma (MPM) is poor and median survival is about 12 months. If any associations can be established between biomarkers and MPM, there will be benefit to clinical practice. In this study, the aim is to examine expression levels of the genes selected from relevant literature and utilizing in silico methods in the determination of prognosis of MPM. The study group consisted of 54 MPM patients treated by chemotherapy. The expression of 6 genes; sestrin 1 (SESN1), laminin subunit alpha 4 (LAMA4), midkine (MKN), fibulin-3 (EFFL-3), syndecan-1 (SOC-1) and hyaluronan-2 (HYA-2) were examined by qPCR in the tumor tissues. SESN1 and LAMA4 were identified using an in house R based script “Unsupervised Survival Analysis Tool.” MKN, EFFL-3, SOC1 and HYA-2 were determined according to existing literature. We used two housekeeping genes; glucose-6-phosphate dehydrogenase (G6PD), TATA-box binding protein (TBP) as controls. qPCR Relative quantification of gene expression was based on the geometric mean G6PD, TBP. The relation between gene expression and prognosis was determined by categorizing samples into two groups using all possible cut-off values, analyzed by the Log Rank test (Log-rank test with multiple cut-offs). Cut-offs generating a p value less than 0.05, between %10-90 percentiles were considered significant. Mean age of study group was 62.5± 9.7 years (r:36-82). Of the patients, 43 (79.6%) had epithelioid cell type mesothelioma. The median survival (MS) for all patients was 10 (±1.18 SE) months (CI 95%;7.69 to 12.30). Twenty-five patients (46.3%) survived less than 12 months, 29 (53.7%) more than 12 months, and 4 (7.4%) were still surviving at the end of the study. The clinical factors that was associated with survival were histopathology (p=0.004) and stage (p=0.036). MKN, EFFL-3, SOC1, HYA-2, SESN-1 were found to be associated with survival time by univariate analyses. After the correction with histopathology and stage, MKN (p=0.041), SOC1 (p=0.015), HYA-2 (p=0.003), SESN-1 (p=0.028) were found to be related with survival time. Additionally, in only epithelioid type MPM patients, HYA-2 expression was found to be related with survival time according to multivariate analyses (p=0.016). High MKN expression is potential biomarker of poor prognosis and high SOC1, HYA-2, SESN-1 expressions are potential biomarkers of good prognosis in MPM patients, and should be further investigated. High HYA-2 expression also can be utilized as good prognosis biomarker for epithelioid type MPM. *This study was partly supported by General Directorate of Health Researches, Republic of Turkey, Ministry of Health.

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues.

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

Abstract 12: c-MYC, EGFR, and FGFR2 expression and response and survival in neoadjuvant treated gastric cancer

Abstract Introduction: Based on high-throughput transcriptional profiling and array comparative genomic hybridization, a three-gene predictor encompassing c-MYC, EGFR and FGFR2 has been identified to predict survival in cisplatin and fluorouracil (FU) – treated metastatic gastric cancer (GC) patients by others (1). The aim of our study was to determine the value of the expression of these three genes to predict response and/or survival for GC patients treated by a platin/5FU based chemotherapy in the neoadjuvant setting. Material and methods: Expression was analyzed in pretherapeutic tumor biopsies of 69 patients treated by platinum/5FU based neoadjuvant chemotherapy. Histopathological response was divided into three grades, tumor regression grade 1 (TRG1) corresponding to complete/major response, TRG2 corresponding to partial and TRG3 corresponding to minimal or no response. mRNA was isolated from manually microdissected formalin fixed and paraffin embedded tumor biopsies. Relative quantification of gene expression was performed by real time PCR using the delta-delta Ct method. As reference genes IPO8 and POLR2A were used and normalization was based on geometric averaging. Analyses were done in triplicates and the mean value was calculated. Fisher`s exact test or χ2-test (two sided) were used to test for an association with response. Overall survival was analyzed by log rank tests and Cox regression analyses. Results: Performing dichotomization of the patients based on median gene expression levels revealed a significant association of high c-MYC expression with minimal or no response (TRG3) (p=0.013). No other associations with response were observed for EGFR or FGFR2 expression in the study as a whole or in a separate analysis of patients with intestinal and nonintestinal type tumors. Testing various combinations of the expression of the three genes did not reveal a stronger association with response when compared to c-MYC expression alone. In the study as a whole, no statistically significant associations with overall survival were found. In the subgroup of nonintestinal-type tumors, univariate Cox regression analysis revealed a significantly increased risk of death for patients with a high expression of c-MYC (HR 59.56, 95%CI 1.28-2776.3; p=0.037). Conclusion: Our results indicate a prominent role of c-MYC expression to predict tumor shrinkage after platin/5FU-based neoadjuvant chemotherapy in GC and they suggest a prognostic relevance of this gene in particular for patients with nonintestinal-type gastric carcinomas. Reference: 1. Kim HK et al.: The Pharmacogenomics Journal (2012) 12: 119-127. Citation Format: Anna Munzig, Lukas Bauer, Julia Slotta-Huspenina, Alexander Novotny, Karen Becker, Alexander Hapfelmeier, Gisela Keller. c-MYC, EGFR, and FGFR2 expression and response and survival in neoadjuvant treated gastric cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 12.

Agrobacterium-mediated in-planta transformation of rough lemon (Citrus jambhiriLush.)

Agrobacterium-mediated in-planta genetic transformation of rough lemon (Citrus jambhiri Lush.) circumventing the need of tissue culture is reported. Mature seeds of rough lemon with removed integument were germinated on moist filter paper and the seeds with emerged plumule (1–2 cm) were pricked at the stem part using a sterile needle followed by immersing in Agrobacterium suspension carrying antifungal β-1,3-glucanase gene under the control of CaMV 35S promoter and NOS terminator. The Agrobacterium-infected seedlings were blot dried and transferred to sowing trays containing field soil + farm-yard manure (3:1) and moved to transgenic glass house for growth of the seedlings. The genomic DNA of T0 seedlings (142) was PCR analysed using gene specific PCR primers leading to identification of eight seedlings (5.63%) carrying β-1,3-glucanase transgene. Transcription of the transgene in eight putative transformants was determined by RT-PCR and quantification of relative gene expression by qRT-PCR revealed upto 4.5-fold increase in seedling number 41, as compared to non-transgenic control.

Appropriate real-time PCR reference genes for fluoride treatment studies performed in vitro or in vivo

ObjectiveQuantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo. DesignFive candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5mM sodium fluoride (NaF) for 6 or 18h followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples. ResultsThe most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene. ConclusionsThis study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ.

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-β and β-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.