scholarly journals Expression level of DREB gene of local corn cultivars from Kisar Island-Maluku, Indonesia, using quantitative real time polymerase chain reaction

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Hermalina Sinay ◽  
Estri Laras Arumingtyas
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Flag Leaf ◽  
Block Design ◽  
Rna Extraction ◽  
Expression Level ◽  
Total Rna ◽  
Deep Yellow ◽  
Dreb Gene

The research objective was to determine the expression level of dehydration responsive element binding (DREB) gene of local corn cultivars from Kisar Island Maluku, Indonesia. The study was designed as randomized block design with single factor consist of six local corn cultivars obtained from farmers in Kisar Island and one reference varieties which has been released as a drought-tolerant varieties and obtained from Cereal Crops Research Institute Maros South Sulawesi. Isolation of total RNA from the second leaf after the flag leaf at the 65 days after planting were carried out according to the protocols of the R and ABlueTM Total RNA Extraction Kit, and was used as a template for cDNA synthesis. Amplification of cDNA from total RNA was carried out according to the protocol of One-Step Reverse Transcriptase PCR Premix Kit. Real Time-PCR was performed usingcDNA from reverse transcription following the procedures of Real MODTM Green Real-Time PCR Master Mix Kit. The real time-PCR data were analyzed using relative quantification method based on the critical point/cycle threshold. The highest DREB gene expression was showed by Deep Yellow local corn cultivar, and the lowest one was showed by Rubby Brown Cob cultivar. The DREB gene expression level of deep yellow local corn cultivar was even higher than Srikandi variety as a reference variety.

Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4401-4401
Author(s):  
Ebrahim Sakhinia ◽  
Mahboubeh Farahangpour ◽  
John A. Liu Yin ◽  
Gerard Brady ◽  
Judith A. Hoyland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Cyclin D3 ◽  
Expression Level ◽  
Tumor Type ◽  
Gene Signatures ◽  
Significant Difference ◽  
The Mean ◽  
Microarray Gene

Abstract Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.

scholarly journals Evaluation of GNMT Gene Expression in Prostate Cancer Tissues using Real-Time PCR

2021 ◽  
Author(s):  
Niloofar Dehghani ◽  
Masoud Salehipour ◽  
Babak Javanmard
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Benign Prostatic Hyperplasia ◽  
Real Time ◽  
Real Time Pcr ◽  
Prostatic Hyperplasia ◽  
Expression Level ◽  
P Value ◽  
Cancer Tissue ◽  
Tissue Samples

Introduction: Prostate cancer is the second most common cancer and the leading cause of cancer-related deaths worldwide. In the present study, the expression level of glycine N-methyl transferase gene (GNMT) was investigated in prostate cancer tissue. The GNMT enzyme is encoded by the GNMT gene. Increased GNMT gene expression increases the conversion of glycine to sarcosine and results in the elevated levels of sarcosine in blood and urine. Methods: The expression level of GNMT gene in tissue samples of patients with prostate cancer was compared with those with benign prostatic hyperplasia using Real-Time PCR technique. Results: The GNMT gene expression level increased significantly in prostate cancer patients compared with those with benign prostatic hyperplasia (p-value <0.001). In addition, the expression level of GNMT gene was stage-dependent and  significant increases were observed in all stages of prostate cancer compared with those with benign prostatic hyperplasia (p-value <0.001). Conclusion: The concentration of sarcosine is controlled by GNMT and it seems that increasing the expression level of GNMT gene increases the level of sarcosine concentration. Thus, it appears that increased levels of GNMT expression occur in the early stages of prostate cancer. Therefore, periodic measurement of GNMT expression levels can detect prostate cancer before it forms a cancer cell and invades other tissues.

scholarly journals Co Expression of GMFβ, IL33, CCL2 and SDF1 Genes in the Acute Stage of Toxoplasmosis in Mice Model and Relation for Neuronal Impairment

2021 ◽  
Author(s):  
Mehri Najafi ◽  
Razieh Amini ◽  
Amir Hossein Maghsood ◽  
Mohammad Fallah ◽  
Faeze Foroughi-Parvar
Keyword(s):  
Gene Expression ◽  
Neurodegenerative Diseases ◽  
Real Time ◽  
Real Time Pcr ◽  
Early Stage ◽  
Rna Extraction ◽  
Acute Stage ◽  
Mice Model ◽  
Toxoplasma Infection ◽  
Inflammatory Protein

Background: Toxoplasma gondii is an obligate intracellular parasite that migrates through macrophages or dendritic cells to neurons and nerve cells. Glia Maturation Factor (GMF) is a pre-inflammatory protein that is expressed in the central nervous system (CNS). GMFβ expression is related to IL33 and CCL2 and SDF1 in some neurodegenerative diseases. According to the importance of GMFβ in neurodegenerative diseases and its association with IL33, CCL2 and SDF1 genes, this study was designed to determine the level of expression of these genes in the brains of mice with acute toxoplasmosis. Methods: Tachyzoites of T. gondii RH strains were injected to 5 Swiss Albino mice. At the same time, healthy mice were inoculated with the Phosphate-buffered saline (PBS). Their brains were removed and kept at -70 oC in order to RNA extraction, cDNA syntheses and Real Time PCR performance. The level of gene expression was investigated with SYBR Green Quantitative Real-Time PCR. Results: GMFβ gene expression increased significantly (P=0.003) 3.26 fold in Toxoplasma infected mice in comparison to the control. GMFβ gene expression was associated with increased expression level of IL33, CCL2, and SDF1 genes. Conclusion: Considering the prominent role of GMFβ in CNS as well as the immune system, the elevation of GMFβ, IL33, CCL2 and SDF1 genes expression in the early stage of toxoplasmosis is associated with the occurrence of neuropathological alterations. Detection of these genes as an indication of brain damage in the early stages of Toxoplasma infection can prevent neurodegenerative disorders following acquired toxoplasmosis.

scholarly journals Lead and cadmium influences on the metallothionein expression level in Lymnaea stagnalis L. adults

1970 ◽  
pp. 281-285
Author(s):  
S. E. Dromashko ◽  
S. N. Shevtsova ◽  
A. S. Babenko
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Test System ◽  
Expression Level ◽  
Pond Snail ◽  
Cadmium Ions ◽  
Lead And Cadmium ◽  
Expression Evaluation

Aim. The article deals with the assessment of some heavy metals effects on metallothionein expression in such a test system as the great pond snail L. stagnalis. Methods. There are methods of mollusk specimen cultivation and gene expression level estimation (PCR in real-time mode), as well as data analysis. Results. A significant increase in MT gene expression was observed in both young (4.5 months) and older (7 months) mollusks on the 7th day of exposure to 0.003 mg/L of lead ions as well as significant increase in MT gene expression was observed in older mollusks on the 7th day of exposure to 0.0001–0.001 mg/L of cadmium ions. Conclusions. The expediency of L. stagnalis adults using at the age of 3 to 5 months with other test systems for biotesting liquid metal-containing waste based on the gene expression evaluation in a short (no more than 7 day) experiment using the Real Time PCR has been shown.Keywords: Lymnaea stagnalis, lead/cadmium, metallothioneins, gene expression, real time PCR.

scholarly journals P0069MICRO RNA 155, 210, 494, 29B AND 34A EXPRESSION PROFILE IN PREECLAMPSIA AND NORMAL PREGNANCIES

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Ali Ghafari ◽  
Mahboob Lessan pezeshki ◽  
Mojtaba Saffari
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Consensus Sequence ◽  
Rna Extraction ◽  
Micro Rna ◽  
Control Group ◽  
Transcriptional Level ◽  
Total Rna ◽  
Qrt Pcr ◽  
Micro Rnas

Abstract Background and Aims Preeclampsia (PE) is a complex disorder that is characterized by hypertension and proteinuria after the 20th week of pregnancy, and it causes most neonatal morbidity and perinatal mortality. Despite many efforts, the knowledge acquired regarding its pathogenesis and pathophysiology does not allow us to treat it efficiently. It is not possible to arrest its progressive nature, and the available therapies are limited to symptomatic treatment. MicroRNAs (miRNAs) are small non-coding RNAs of approximately 19–23 nucleotides that can bind to the 3’ untranslated region of target mRNAs resulting in the degradation and translation inhibition of the mRNA, thereby regulating gene expression at the post-transcriptional level. Many studies have confirmed deregulated miRNA in pregnant patients with PE, and the function and mechanism of these differentially expressed miRNA are gradually being revealed. Method Sample collection Among the patients in the maternity ward, Department of Obstetrics and Gynecology. pregnant women who carried fetuses from 26 to 40 weeks of gestation were selected for study. Plasma samples were obtained from a total of 90 women in two groups; 48 being preeclamptic and 42 being healthy pregnancies, forming the control group. None of these subjects had undergone an invasive procedure. A volume of 5 mL maternal venous blood was drawn and collected in an ethylenediamine tetraacetic acid (EDTA) tube. The blood samples were centrifuged initially at 1200 g for 10 min and a second time at 10,000 g for 10 min, and 500 mL from the supernatant layer were used. The supernatant layer of the plasma was taken to Department of Molecular Biology and Genetics, Molecular Diagnosis Laboratory for storage at -80°C until the time of study. Total RNA extraction Total RNA was isolated from plasma using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer protocol. Total RNA was analyzed using the commercially available Bioanalyzer Agilent RNA 6000 picoassay. MicroRNA isolation from maternal plasma MicroRNAs were obtained with the High-Specificity miRNA QRT-PCR detection kit (Stratagene-an Agilent Technologies Company, USA-Canada) according to manufacturer recommendations. Micro RNAs were subjected to a polyadenylation reaction as previously described. Next, using an oligo dT primer harboring a consensus sequence, reverse transcription was performed using an Affinity Script RT/RNase Block Enzyme mixture (Stratagene). miRNA was anayzed using the Bioanalyzer 2100- Agilent Small RNA assay. QRT-PCR platform The cDNA was amplified by real-time PCR. The real-time PCR analysis was performed on a Stratagene Mx3005P. This reaction contained a microRNA-specific forward primer, a TaqMan probe complementary to the 3ꞌ of the specific microRNA sequence, as well as part of the polyA adaptor sequence, and a universal reverse primer complementary to the consensus 3ꞌ sequence of the oligodT tail. Results were recorded in clinic sheets and were analyzed using SPss version 22. Results G1 consist of 48 and G2 42 pts. There were no differences in two group in respect to age (mean age was 28± 9.5 and 27± 5.23 in G1 and G2 respectively). Micro RNA155, 210 and 494 were significantly higher in plasma of G1 (Pv 0.001, 0.0001, 0.005 respectively). Micro RNA 29b was significantly lower in G1 (Pv 003). And there was no difference between two groups in respect to Micro RNA 34a (Pv 0.09). And also PE patients with higher plasma creatinine have more plasma level of Micro RNA155 and 210. There was a significant correlation between Micro RNA 494 and anemia in PE patients Conclusion In this study we showed that Micro RNA155, 210 and 494 increased and Micro RNA 29b decreased in PE and after clarifying by further studies may use as a predictor in clinical practice.

scholarly journals Recruitment of cyanobacteria by reverse transcription quantitative real-time PCR based on expression of Microcystis gene

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7188
Author(s):  
Long Yu ◽  
Xiaofei Wu ◽  
Yang Yu ◽  
Limei Shi ◽  
Min Zhang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lake Taihu ◽  
Rna Extraction ◽  
Sybr Green ◽  
Quantitative Detection ◽  
Control Group ◽  
Quantitative Real Time Pcr ◽  
Total Rna ◽  
Pcr Method

In this study, a SYBR Green quantitative real-time PCR method was established and applied. Relative expression of the synthetic genes from Microcystis gas vesicles (gvpC), algal toxin genes (mcyA), and polysaccharides (espL) from water and sediments of Meiliang Bay and from the center of Lake Taihu were tested from January to June, 2017. Indoor Microcystis aeruginosa was used as the control group. The kit for total RNA extraction in Microcystis was optimized. Results showed that the optimized kit extracted high-concentrations and high-quality total RNA from Microcystis. The extraction purity and concentration were significantly higher than those extracted by the original kit. The transcription level of gvpC increased gradually until a peak was reached in March. However, expression of gvpC decreased continuously at the proliferating and floating stages of Cyanobacterial biomass. The maximum level of expression of gvpC in April in comparison to expression of mcyA in March occurred first. We found that the SYBR Green qRT-PCR method, which is characterized by high specificity, repeatability, is rapid, and can be used for quantitative detection of expression of gvpC, mcyA, and espL. The recruitment of cyanobacteria is the process in which cyanobacteria in the sediment began to regain their activity, started to grow and migrated to the water column.

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