Development of a Multiplex Assay for Rapid Assessment of Gene Expression Profiling of Rho GTPases Family Genes In MDS Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2907-2907
Author(s):  
Xuejun Shao ◽  
Meihua Miao ◽  
Zi-Xing Chen
Keyword(s):  
Gene Expression ◽  
Expression Profiling ◽  
Rho Gtpase ◽  
Rapid Assessment ◽  
Multiplex Assay ◽  
Expression Level ◽  
Control Group ◽  
Qrt Pcr ◽  
Flow Cytometric ◽  
Significant Difference

Abstract Abstract 2907 We have previously identified a panel of genes by gene expression profiling which differentially expressed between primary bone marrow CD34+ cell of MDS patients and the normal control cells. The expression pattern of these genes were also confirmed individually by qRT-PCR in expanded cohort of MDS patients. To further explore the feasibility of using proper number of these genes as biomarkers and evaluate the clinical significance in the diagnosis and prognostic prediction of MDS patients, a convenient, robust, sensitive, specific and less expensive method need to be developed. Therefore, endeavors have been made to establish a bead-based flow-cytometric multiplex assay. To establish this method, a technique called Multiplex ligation-dependent probe amplification (MLPA) was employed to amplify several targeted cDNAs by using single pair of identical primers. Each MLPA probe consists of two short synthetic oligonucleotide, and the tag which was coupled chemically to the fluorescent beads was complementary to one probe. Just as a pilot study to simultaneously detect the expression of four genes of the Rho GTPase family, five beads with different fluorescence intensity coupled to RAC2, RhoBTB3, SPA-1, Rap1GAP and GAPDH were designed. Biotinylated PCR amplicons were then hybridized to the complementary tag on each bead set. Bound amplicons were then detected by flow cytometry using a streptavidin-linked reporter dye, PE. 111 BM specimens were analyzed in total, consisting of RA(22), RAEB(22), RAEBt(9), AML(33), and control group (22, including hyperplastic anemia, iron deficiency anemia, aplastic anemia etc). The difference in the transcriptional level of RAC2, RhoBTB3, SPA-1 and Rap1GAP relative to GAPDH were analyzed using wilcoxon non-parametric test and SNK method among different groups. The results were confirmed by qRT-PCR. The bead-based flow-cytometric array had a excellent sensitivity and a wide linear range, could get a positive signal for PCR product from 0.0025 to 0.1umol, the fine specificity was proved by no cross-hybridization signals presented among different bead set, and the reproducibility were also good enough(P<0.001). the expression profiling of RAC2, RhoBTB3, SPA-1,Rap1GAP and GAPDH detected by this liquid bead-based flow-cytometric array were thus obtained. A significant difference among five groups for the expression level of RAC2, RhoBTB3, SPA-1 and Rap1GAP relative to GAPDH was found (P<0.0001, P=0.0491, P=0.0206 and P=0.0046 respectively). For RAC2, no difference existed between AML and control groups, while both these two groups demonstrated significant difference compared with RA, RAEB and REABt groups. The RA group had the highest expression level, AML the lowest; and RAEB in between. For RhoBTB3, a significant difference could be seen between RAEBt and other groups, RAEBt had the highest level of RhoBTB3. There was no difference in SPA-1 expression level determined between each two groups. However, an increasing tendency from control group to RA, RAEB, and REABt could be observed, whereas the expression of SPA-1 dramatically dropped to the lowest level in AML. For Rap1GAP, differences were found between AML and other group; These results were validated by qRT-PCR, and the data obtained by each method had close linear correlation, the Pearson correlation coefficient was 0.930, 0.946, 0.945 and 0.921 for RAC2, RhoBTB3, SPA-1 and Rap1GAP respectively (P<0.001 for all four). Based on these preliminary results, A liquid bead-based flow-cytometric multiplex assay for a rapid assessment of gene expression profile had been successfully developed and validated by qRT-PCR. This method will facilitates the clinical usage of a certain group of Rho GTPase family genes as well as other appropriate genes as useful biomarkers in the diagnosis of MDS patients and provide interesting clues to further explore the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of mannuronic acid (M2000) on gene expression profile of signal transducer and activator of transcription proteins (STATs) in rheumatoid arthritis patients

Reumatismo ◽  
2020 ◽  
Vol 72 (2) ◽  
pp. 93-102
Author(s):  
N.A.G. Gaafar ◽  
M. Aslani ◽  
Z. Aghazadeh ◽  
S.S. Mortazavi-Jahromi ◽  
A. Razavi ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Conventional Therapy ◽  
Gene Expression Level ◽  
Expression Level ◽  
Control Group ◽  
Healthy Controls ◽  
Signal Transducer ◽  
Mannuronic Acid ◽  
Significant Difference

Rheumatoid arthritis (RA), a form of inflammatory arthritis, is a chronic joint disease characterized by pain and inflammation that affects 0.5% to 1% of the population worldwide. The safety, efficacy, tolerability, and potency of β-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property has been reported by several in vitro studies, experimental models and clinical trials phase I/II and III in ankylosing spondylitis and rheumatoid arthritis (RA) patients This research is designed to study the therapeutic efficacy of oral administration of mannuronic acid in RA patients who had inadequate response to conventional drugs and to assess the effect of this drug on gene expression of the signal transducer and activator of transcription (STATs) protein (STAT1, STAT3, STAT4, and STAT6). The study has included 15 RA patients who had an insufficient response to the conventional therapy. The oral dose of mannuronic acid was 1000mg divided into two 500 mg doses per day for 3 months as an addition to conventional therapy. There were 15 healthy volunteer in the control group. Blood samples were collected from both groups, once from healthy controls and twice from RA patients before and after treatment by M2000. The peripheral blood mononuclear cells (PBMCs) were isolated to assess the gene expression level of STAT1, STAT3, STAT4, and STAT6 using the real-time PCR method. Results obtained in this study demonstrated a significant difference in the gene expression level of STAT1 between healthy controls and patients before treatment as well as a significant reduction in RA patients after treatment compared with the level before treatment. In addition, the gene expression level of STAT3 and STAT4 showed a significant reduction in RA patients after treatment compared to patients before treatment, while there was no significant difference between RA patients before treatment and the healthy control group for both molecules. On the other hand, there was no change in the gene expression level of STAT6 among all groups. The outcomes of this study confirmed that β-D-mannuronic acid (M2000) has the ability to control the levels of STAT1, STAT3 and STAT4 in RA patients, and might be beneficial in the management and therapy of RA.

scholarly journals Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients

2021 ◽  
Author(s):  
Frank Faltraco ◽  
Denise Palm ◽  
Adriana Uzoni ◽  
Lena Borchert ◽  
Frederick Simon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dermal Fibroblast ◽  
Sleep Onset ◽  
Dermal Fibroblasts ◽  
Control Group ◽  
Adhd Group ◽  
Healthy Controls ◽  
Circadian Gene ◽  
Significant Difference ◽  
Circadian Preference

AbstractA link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

scholarly journals Effects of L-655,708 on expression changes of GABA, glutamate, and beta-endorphin induced by propofol anesthesia in rats

2018 ◽  
Vol 16 ◽  
pp. 205873921879670
Author(s):  
Jin Wang ◽  
Xinyi Li ◽  
Huisheng Wu ◽  
Jianjuan Ke ◽  
Zongze Zhang ◽  
...  
Keyword(s):  
Dentate Gyrus ◽  
Cognitive Dysfunction ◽  
Sham Group ◽  
Postoperative Cognitive Dysfunction ◽  
Expression Level ◽  
Control Group ◽  
Beta Endorphin ◽  
Anesthetized Rats ◽  
Significant Difference ◽  
Dentate Gyrus Region

Anesthetics are considered to be one of the important inducing factors of postoperative cognitive dysfunction (POCD). The hippocampal region of the rat is one of the action sites of general anesthesia drugs. L 655,708, a reverse agonist of gamma aminobutyric acid (GABA) receptor, can significantly improve short-term memory dysfunction in mice after anesthetized with isoflurane. So the purpose of this study is to investigate the effects of L-655,708 on expression of GABA, glutamate (GLU), and beta-endorphin (β-EP) in the dentate gyrus region of the hippocampus and cognition of rats anesthetized with propofol. In all, 30 male Sprague–Dawley (SD) rats were randomly allocated into the control group, sham group, and L-655,708 group, with 10 in each group. The cognitive function of rats was measured by Morris water maze before and 1 h after administration. Then the rats were sacrificed for brain tissues. Immunohistochemistry was used to study the expression of GABA, GLU, and β-EP in the hippocampus of anesthetized rats in each group. Compared with the control group, the latency of the sham group and L-655,708 group were significantly prolonged after administration ( P < 0.05). However, L-655,708 could shorten the prolonged latency ( P < 0.05). There was no significant difference in times of accessing original platform area between the three groups before and after medication ( P > 0.05). The expression level of GABA in the dentate gyrus region of hippocampus of rats in the sham group was significantly higher than that in the control group ( P < 0.05), while the expression level in the L-655,708 group was significantly lower than that in the sham group ( P < 0.05). No significant difference was found in the expression of GLU in the dentate gyrus region of hippocampus of rats in each group ( P > 0.05). Compared with the control group, the expression of β-EP was significantly lower in the dentate gyrus region of the hippocampus of sham group rats ( P < 0.05). However, the expression of β-EP in the L-655,708 group was significantly higher than that in the sham group ( P < 0.05). Cognitive dysfunction in rats anesthetized with propofol may be related to high expression of GABA and low expression of β-EP in the hippocampus. The mechanism of L-655,708 in reducing the cognitive impairment in propofol anesthetized rats may be bound up with down-regulating the expression of GABA and increasing the expression of β-EP in the hippocampus.

scholarly journals Cholesterol Transporters ABCA1 and ABCG1 Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Metabolic Syndrome

Cholesterol ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Zahra Tavoosi ◽  
Hemen Moradi-Sardareh ◽  
Massoud Saidijam ◽  
Reza Yadegarazari ◽  
Shiva Borzuei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Syndrome ◽  
Mononuclear Cells ◽  
Fasting Blood Glucose ◽  
Control Group ◽  
Regulation Mechanism ◽  
Healthy Individuals ◽  
Peripheral Blood Mononuclear ◽  
Significant Difference ◽  
Abca1 Expression

ABCA1 and ABCG1 genes encode the cholesterol transporter proteins that play a key role in cholesterol and phospholipids homeostasis. This study was aimed at evaluating and comparing ABCA1 and ABCG1 genes expression in metabolic syndrome patients and healthy individuals. This case-control study was performed on 36 patients with metabolic syndrome and the same number of healthy individuals in Hamadan (west of Iran) during 2013-2014. Total RNA was extracted from mononuclear cells and purified using RNeasy Mini Kit column. The expression of ABCA1 and ABCG1 genes was performed by qRT-PCR. Lipid profile and fasting blood glucose were measured using colorimetric procedures. ABCG1 expression in metabolic syndrome patients was significantly lower (about 75%) compared to that of control group, while for ABCA1 expression, there was no significant difference between the two studied groups. Comparison of other parameters such as HDL-C, FBS, BMI, waist circumference, and systolic and diastolic blood pressure between metabolic syndrome patients and healthy individuals showed significant differences (P<0.05). Decrease in ABCG1 expression in metabolic syndrome patients compared to healthy individuals suggests that hyperglycemia, related metabolites, and hyperlipidemia over the transporter capacity resulted in decreased expression of ABCG1. Absence of a significant change in ABCA1 gene expression between two groups can indicate a different regulation mechanism for ABCA1 expression.

scholarly journals Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

2020 ◽  
Author(s):  
Kwan-Sik Min ◽  
Jong-Ju Park ◽  
So-Yun Lee ◽  
Munkhzaya Byambaragchaa ◽  
Myung-Hwa Kang
Keyword(s):  
Gene Expression ◽  
Chorionic Gonadotropin ◽  
Expression Profiles ◽  
Expression System ◽  
Laboratory Animals ◽  
Metabolic Clearance ◽  
Qrt Pcr ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin

Abstract Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).Results: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.Conclusions: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

scholarly journals qRT-PCR-based DNA homologous recombination associated 4-gene score predicts pathologic complete response to platinum-based neoadjuvant chemotherapy in triple-negative breast cancer

2021 ◽  
Author(s):  
Ke Zuo ◽  
Xiaoying Yuan ◽  
Xizi Liang ◽  
Xiangjie Sun ◽  
Shujin Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Homologous Recombination ◽  
Neoadjuvant Chemotherapy ◽  
Triple Negative ◽  
Pathologic Complete Response ◽  
Complete Response ◽  
Expression Level ◽  
Cancer Center ◽  
Gene Score ◽  
Qrt Pcr

Abstract PurposeCumulative evidences suggested the addition of platinum agents as neoadjuvant chemotherapy (NACT) could improve pathologic complete response (pCR) in triple-negative breast cancers (TNBC). We tried to develop a DNA homologous recombination (HR) associated gene expression score to predict tumor sensitivity to platinum-based NACT in TNBC.MethodsA retrospective cohort of 127 TNBC patients, who were diagnosed and received platinum-based NACT in Fudan University Shanghai Cancer Center from 2012 to 2017, was included in this study. By using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the expression level of eight HR associated genes was analyzed from the formalin-fixed paraffin-embedded core needle biopsy samples which obtained before NACT. A random forest model was built to estimate the weight of each gene expression level and clinical-pathological factors. The training set was used to modulate parameters and select the best model. The performance of the final model was evaluated in the validation set. ResultsA 4-gene (BRCA1, XRCC5, PARP1, RAD51) expression scoring system was developed. TNBC with higher score had nearly quadruple likelihood to achieve pCR to platinum-based NACT compared with a lower score [odds ratio (OR)=3.878; P<0.001]. At the cut-off value of -2.644, the 4-gene score system showed high sensitivity in predicting pCR in breast (93.0%) and pCR in both breast/axilla (91.8%), while, at the cut-off value of -1.969, the 4-gene score showed high specificity for pCR in breast (85.7%) and pCR in both breast/axilla (80.8%). ConclusionThe qRT-PCR-based 4-gene score has the potential to predict pCR to platinum-based NACT in TNBC.

Investigation of TAp63 gene Expression and Follicle Count Using Melatonin in Cisplatin-induced Ovarian Toxicity

2020 ◽  
Author(s):  
Hacı Öztürk Şahin ◽  
Mehmet Nuri Duran ◽  
Fatma Sılan ◽  
Ece Sılan ◽  
Duygu Sıddıkoglu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Female Rats ◽  
Histological Evaluation ◽  
Saline Control ◽  
Control Group ◽  
Ovarian Toxicity ◽  
Significant Difference ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

Abstract Background: Premature ovarian failure is among the most important side effects of chemotherapy during reproductive period. Preserving ovarian function is gradually gaining importance during oncologic treatment. The present study aims to investigate the potential of melatonin to protect from cisplatin-induced ovarian toxicity in rats. Twenty nine female rats were divided to three groups: Saline control group (Group 1), cisplatin group (Group 2), and cisplatin+melatonin group (Group 3). While the rats in Groups 2 and 3 were administered 5 mg/kg single dose of cisplatin via intra-peritoneal (IP) route, the rats in Group 3 were started on melatonin (20 mg/kg IP) before cisplatin administration and continued during 3 consecutive days. Ovaries were removed one week after cisplatin administration in all groups. Blood samples were obtained before the rats were decapited. Histological evaluation, follicle count, and classification were performed. TAp63 mRNA expression was evaluated using mRNA extraction and real-time polymerase chain reaction (PCR) method. Serum estradiol (E2) and anti-mullerian hormone (AMH) values were measured with enzyme immune-assay technology. Results: While primordial follicles were seen to decrease in Group 2 as compared to Group 1 (p:0.023), primordial follicle count was observed to be preserved significantly in melatonin group as compared to Group 2 (p:0.047). Moreover, cisplatin-induced histo-pathological morphology was preserved in favor of normal histology in melatonin group. A significant difference was not observed between groups with regard to mean serum AMH and E2 values (p:0.102 and p:0.411, respectively). While TAp63 gene expression significantly increased in Group 2 as compared to control group (p:0.001), we did not detect a statistically significant difference in cisplatin+melatonin group, although gene expression decreased (p:0.34). Conclusion: We conclude that concurrent administration of melatonin and cisplatin may protect from ovarian damage.

scholarly journals Effect of acute noise trauma on the gene expression profile of the hippocampus

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Chang Ho Lee ◽  
Kyung Woon Kim ◽  
So Min Lee ◽  
So Young Kim
Keyword(s):  
Gene Expression ◽  
Noise Exposure ◽  
Control Group ◽  
Sprague Dawley ◽  
Qrt Pcr ◽  
Neuronal Systems ◽  
Immune Related Genes ◽  
Pathway Analyses ◽  
Upregulated Genes ◽  
External Stimuli

Abstract Background This study aimed to investigate the changes in the expression of hippocampal genes upon acute noise exposure. Methods Three-week-old Sprague–Dawley rats were assigned to control (n = 15) and noise (n = 15) groups. White noise (2–20 kHz, 115 dB sound pressure level [SPL]) was delivered for 4 h per day for 3 days to the noise group. All rats were sacrificed on the last day of noise exposure, and gene expression in the hippocampus was analyzed using a microarray. Pathway analyses were conducted for genes that showed differential expression ≥ 1.5-fold and P ≤ 0.05 compared to the control group. The genes included in the putative pathways were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results Thirty-eight upregulated genes and 81 downregulated genes were identified. The pathway analyses revealed that upregulated genes were involved in the cellular responses to external stimuli and immune system pathways. qRT-PCR confirmed the upregulation of the involved genes. The downregulated genes were involved in neuronal systems and synapse-related pathways, and qRT-PCR confirmed the downregulation of the involved genes. Conclusions Acute noise exposure upregulated the expression of immune-related genes and downregulated the expression of neurotransmission-related genes in the hippocampus.

scholarly journals Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy

2017 ◽  
Vol 23 (5) ◽  
pp. 440-448 ◽  
Author(s):  
Reza Gholamnezhadjafari ◽  
Nader Tajik ◽  
Reza Falak ◽  
Reza Aflatoonian ◽  
Sanaz Dehghan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Inflammatory Factors ◽  
Control Group ◽  
Organ Donors ◽  
Complement Components ◽  
Tnf Α ◽  
Brain Dead

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

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