Real Time PCR Using Molecular Beacons: A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis

2003 ◽  
pp. 295-310 ◽  
Author(s):  
Riccardo Manganelli ◽  
Sanjay Tyagi ◽  
Issar Smith
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Point Mutations ◽  
Molecular Beacons

Molecular Beacons for Sensitive Gene Detection in Living Cells

2003 ◽  
Author(s):  
Andrew Tsurkas ◽  
Gang Bao
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Molecular Beacon ◽  
Point Mutations ◽  
Living Cells ◽  
Molecular Beacons ◽  
High Signal ◽  
Gene Detection ◽  
Real Time Imaging ◽  
Background Ratio

Real-time imaging of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of cancer, including cancer diagnosis and analysis. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA as tumor markers due to their high signal-to-background ratio, and their improved specificity in detecting point mutations. However, the harsh intracellular environment does limit the sensitivity of MB-based gene detection. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, or opened due to non-specific interactions. To overcome this difficulty, we have developed a novel dual FRET molecular beacons approach in which a pair of molecular beacons, one with a donor fluorophore and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of false-positive events. We have performed systematic in-solution and cellular studies of dual FRET molecular beacon and demonstrated that this new approach allows for real-time imaging of gene expression in living cells.

Respon Morfologis dan Ekspresi Gen Aquaporin pada Padi IR 64 yang Mengalami Cekaman Kekeringan pada Fase Reproduktif (Morphological Responses and Aquaporin Gene Expression in Rice IR64 under Drought Stress at the Reproductive Stage)

2018 ◽  
Vol 7 (2) ◽  
Author(s):  
Made Pharmawati ◽  
Ni Nyoman Wirasiti ◽  
Luh Putu Wrasiati
Keyword(s):  
Gene Expression ◽  
Drought Stress ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Plant ◽  
Reproductive Stage ◽  
Limiting Factors ◽  
Rice Grain ◽  
Aquaporin Gene ◽  
Peg Treatment

Abstrak Cekaman kekeringan merupakan faktor pembatas penting bagi pertumbuhan dan produktivitas tanaman termasuk padi.      Penelitian ini bertujuan menganalisis respon padi IR64 terhadap cekaman kekeringan dengan pemberian polietilen glikol (PEG) pada fase reproduktif.  Penelitian juga bertujuan menganalisis ekspresi gen aquaporin akibat cekaman kekeringan.  Bibit padi ditanam dalam pot dan perlakuan PEG dengan konsentrasi 108g/L (-0.25MPa) dan 178g/L (-0.52 MPa) diberikan saat munculnya panikula. Perlakuan diberikan selama 2 minggu, kemudian tanaman disiram kembali.  Ekspresi gen diamati pada akhir perlakuan dengan semi kuantitatif real time PCR.  Ekstraksi RNA menggunakan RNeasy plant mini kit, sedangkan sintesis cDNA menggunakan Transcriptor First Strand cDNA Kit.  Hasil penelitian menunjukkan bahwa jumlah malai dan berat total malai berkurang akibat cekaman kekeringan.  Persentase gabah kosong mencapai 84,6% pada perlakuan PEG-0,52 MPa, sedangkan pada perlakuan PEG -0,25 MPa persentase gabah kosong sebesar 67,8%.  Pada kontrol persentase gabah kosong adalah 10,3%.  Ekspresi gen OsPIP2;7 sedikit menurun pada perlakuan PEG -0,52 MPa.Kata kunci: ekspresi gen, IR64, kekeringan, padi, PEG  Abstract Drought stress is one of the limiting factors of plant growth and productivity including rice.  The aim of this study was to analyze responses of IR64 rice to polyethylene glycol (PEG)-induced-drought stress at the reproductive stage.  This study also aimed to analyze the expression of aquaporin under drought stress.  Rice seedlings were grown in pot system and PEG treatment at concentration of -0.25MPa (108g/L) and -0.52 MPa (178g/L) were given when the panicles arose.  Treatments were conducted for 2 weeks, after that the plants were rewatered.  Gene expression was evaluated at the end of PEG treatment using semi quantitative real time PCR. RNA was extracted using RNeasy plant mini kit, while cDNA synthesis was done using Transcriptor First Strand cDNA Kit.  The results showed that the number and weight of rice ear were less in plant treated with PEG than in control.  The percentage of empty rice grain reached 84.6% at PEG -0.52 MPa, while at PEG -0.25 MPa the percentage of empty grain was 67.8%.  In control plant, the percentage of empty grain was 10.3%.  Drought stress did not alter the expression of OsPIP2;7.  Keywords: drought, gene expression, IR64, PEG, rice

scholarly journals Molecular characterisation of osteoblasts from bone obtained from people of Polynesian and European ancestry undergoing joint replacement surgery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dorit Naot ◽  
Jarome Bentley ◽  
Cluny Macpherson ◽  
Rocco P. Pitto ◽  
Usha Bava ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Joint Replacement ◽  
Real Time Pcr ◽  
S Phase ◽  
European Ancestry ◽  
Mineral Density ◽  
Differentiation Markers ◽  
Joint Replacement Surgery ◽  
Bone Properties

AbstractPopulation studies in Aotearoa New Zealand found higher bone mineral density and lower rate of hip fracture in people of Polynesian ancestry compared to Europeans. We hypothesised that differences in osteoblast proliferation and differentiation contribute to the differences in bone properties between the two groups. Osteoblasts were cultured from bone samples obtained from 30 people of Polynesian ancestry and 25 Europeans who had joint replacement surgeries for osteoarthritis. The fraction of cells in S-phase was determined by flow cytometry, and gene expression was analysed by microarray and real-time PCR. We found no differences in the fraction of osteoblasts in S-phase between the groups. Global gene expression analysis identified 79 differentially expressed genes (fold change > 2, FDR P < 0.1). Analysis of selected genes by real-time PCR found higher expression of COL1A1 and KRT34 in Polynesians, whereas BGLAP, DKK1, NOV, CDH13, EFHD1 and EFNB2 were higher in Europeans (P ≤ 0.01). Osteoblasts from European donors had higher levels of late differentiation markers and genes encoding proteins that inhibit the Wnt signalling pathway. This variability may contribute to the differences in bone properties between people of Polynesian and European ancestry that had been determined in previous studies.

scholarly journals Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0143444 ◽  
Author(s):  
Yong Zhao ◽  
Guilian Li ◽  
Chongyun Sun ◽  
Chao Li ◽  
Xiaochen Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Locked Nucleic Acid ◽  
Pcr Assay ◽  
Locked Nucleic Acid Probe
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