Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs

Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs

Mangan-superoksid-dismutaza (MnSOD) katalizuje NO-zavisno nitrovanje ostatka tirozina

The peroxynitrite-induced nitration of manganese superoxide dismutase (MnSOD) tyrosine residue, which causes enzyme inactivation, is well established, This led to suggestions that MnSOD nitration and inactivation in vivo, detected in various diseases associated with oxidative stress and overproduction of nitric monoxide (NO), conditions which favor peroxynitrite formation, is also caused by peroxynitrite. However, our previous ill vitro study demonstrated that exposure of MnSOD to NO led to NO conversion into nitrosonium (NO+) and nitroxyl (NO-) species, which caused enzyme modifications and inactivation. Here it is reported that MnSOD is tyrosine nitrated upon exposure to NO, as well as that MnSOD nitration contributes to inactivation of the enzyme. Collectively, these observations provide a compelling argument supporting the generation of nitrating species in MnSOD exposed to NO and shed a new light on MnSOD tyrosine nitration and inactivation ill vivo. This may represent a novel mechanism by which MnSOD protects cell from deleterious effects associated with overproduction of NO. However, extensive MnSOD modification and inactivation associated with prolonged exposure to NO will amplify the toxic effects caused by increased cell superoxide and NO levels.Dobro je poznato da peroksinitrit izaziva nitrovanje ostataka tirozina u mangan-superoksid- dismutazi (MnSOD) što dovodi do inaktivacije enzima. Pokazano je da nitrovanje i inaktivacija MnSOD-a nastaje u raznim bolestima za koje je karakteristič an oksidativni stres i povećana produkcija azot-monoksida (NO). Pošto se pri ovim uslovima očekuje nastajanje peroksinitrita predloženo je da peroksinitrit izaziva nitrovanje i inaktivaciju MnSOD in vivo. U našem prethodnom radu pokazali smo da MnSOD katalizuje transformaciju NO u nitrozonijum (NO+) i nitroksil (NO–) reaktivne vrste, te identifikovali neke od modifikacija molekula enzima koje pri tome nastaju izazivajući njegovu inaktivaciju. U ovom radu je pokazano da pri izlaganju MnSOD azot-monoksidu dolazi i do nitrovanja ostatka tirozina u molekulu enzima, što doprinosi njegovoj inaktivaciji. Ovi rezultati ukazuju da pri interakciji MnSOD sa NO dolazi do nastajanja nitrujućih vrsta, što baca novo svetlo na proces nitrovanja ostataka tirozina i inaktivaciju MnSOD in vivo. Ovo može da predstavlja novi mehanizam kojim MnSOD štiti ćeliju odštetnih efekata izazvanih hiperprodukcijom azot-monoksida. Međutim ekstenzivne modifikacije i inaktivacija MnSOD do kojih dolazi pri produženom izlaganju enzima NO, uvećaće toksične efekte izazvane povećanim koncentracijama superoksida i NO u ćeliji

The interaction between salinity stress and seed ageing during germination of Brassica napus seeds

A study was designed to examine the effects of NaCl, high temperature, high relative humidity and storage on the germination of Brassica napus seeds, both when freshly harvested and after natural and accelerated ageing. After exposure to a combination of stressful conditions, the intensity of lipid peroxidation in the seeds, seedling shoot and roots, and seed germination were measured. A significant decline in germination at all concentrations of NaCl indicates that seeds which have been exposed to accelerated ageing are significantly less able to resist even the lowest levels of salt stress. Moreover, the intensity of lipid peroxidation in the seeds and seedlings examined after a year of storage in uncontrolled conditions was higher compared with tests carried out in the year of seed production

Calibration of HPGe Detectors for Environmental Samples Using GEANT4 Simulation

Determination of full energy peak efficiency is one of the most important tasks that have to be performed before gamma spectrometry. Calibration of the measurement system for measuring environmental samples poses a special challenge to the laboratory. Many different approaches to this task have been developed and examined. One of the most detailed is GEANT4, a Monte Carlo simulation toolkit developed for wide variety of applications. The aim of this paper is to apply GEANT4 simulation for calibration of three HPGe detectors, for measurement of aerosol, plant and coal-like environmental samples. The detectors were modeled using the certificate provided by the manufacturer. The samples chosen for the simulation were secondary reference materials produced in the Laboratory for the purpose of experimental calibration. The efficiencies obtained using a simulation were compared with experimental results, in order to evaluate the trueness of the results. Measurement uncertainties for both simulation and experimental values were estimated in order to see whether the results of the realistic measurement fall within acceptable limits.3rd International Conference on Radiation and Applications in Various Fields of Research (RAD), Jun 08-12, 2015, Budva, Montenegr

Calibration of HPGe Detectors for Environmental Samples Using GEANT4 Simulation

Determination of full energy peak efficiency is one of the most important tasks that have to be performed before gamma spectrometry. Calibration of the measurement system for measuring environmental samples poses a special challenge to the laboratory. Many different approaches to this task have been developed and examined. One of the most detailed is GEANT4, a Monte Carlo simulation toolkit developed for wide variety of applications. The aim of this paper is to apply GEANT4 simulation for calibration of three HPGe detectors, for measurement of aerosol, plant and coal-like environmental samples. The detectors were modeled using the certificate provided by the manufacturer. The samples chosen for the simulation were secondary reference materials produced in the Laboratory for the purpose of experimental calibration. The efficiencies obtained using a simulation were compared with experimental results, in order to evaluate the trueness of the results. Measurement uncertainties for both simulation and experimental values were estimated in order to see whether the results of the realistic measurement fall within acceptable limits.3rd International Conference on Radiation and Applications in Various Fields of Research (RAD), Jun 08-12, 2015, Budva, Montenegr

Physico-chemical characteristics of white suger fractions separated by crystal sizes

According to the EU Council Directive 2001/111/EC, related to certain sugars intended for human consumption, a great attention is paid to color in sugar. On the other hand, the non-sucrose compounds having intensive color tend to build into the sucrose crystals. Since Serbia has to work on the new rules concerned sugar quality and harmonization with EU standards, some investigations on sugar crystals are carried out at the request of some sugar factories. Investigations are carried out on sugar crystal size dependence on sugar color and on dissolution rate of different sugar crystal size fractions with the aim to create new sugar products. In this study, sugar samples with different sugar color are divided into 5 fractions by crystal size, defining size distributions. In each fraction the color in solution and the type of color are measured, as well as other relevant physico-chemical parameters. The conclusion is that the sugar color type depends on the crystal size, but sugar crystal solution is not dependent on crystal size except a fraction smaller than 200 µm, which has 30-75% higher sugar color in solution than the other crystal size fractions

Correlation Between Beryllium-7 in Atmospheric Deposit and Ground Level Air in Serbia for 2014 Year

Activity density of beryllium-7 in atmospheric deposit and in ground level air at five monitoring stations (MS Nis, Vranje, Zajecar, Zlatibor, Palic) in Serbia were determined during the period January-December 2014. Activity of cosmogenic radionuclide beryllium-7 was determined on HPG detectors (Canberra, relative efficienty 20%.) by gama spectrometry method. Activity density of beryllium-7 in aerosols were in range 0.5 - 9.8 mBq/m(3) and in deposits were in range 1.8 - 233 Bg/m(2). Based on the obtained results correlation coefficient between aerosols and deposits was calculated and its value ranged from 0.15 to 0.59.3rd International Conference on Radiation and Applications in Various Fields of Research (RAD), Jun 08-12, 2015, Budva, Montenegr

Results from Radionuclide Interlaboratory Comparison in Sediment and Fish

The results of an intercomparison exercise, designed for the determination of anthropogenic and natural radionuclides in sediment and fish samples from the Danube River, are reported. The methods of gross beta and gamma spectrometry measurements were compared. Considering the uncertainties of measurements, a good agreement between the results obtained by two laboratories has been ascertained.3rd International Conference on Radiation and Applications in Various Fields of Research (RAD), Jun 08-12, 2015, Budva, Montenegr

In Vitro Callus Induction from Adult Tissues of Japanese Flowering Cherry Trees and Two Cherry Rootstocks

Several in vitro biotechnological techniques have been developed, all of which require a reliable protocol to produce a responsive callus mass. One of these techniques is callus fusion in vitro, which is reliable for the early detection of (in)compatibility of scions and rootstocks. In this paper, the possibility to obtain friable callus tissues was explored by callus induction of adult tissues of Japanese flowering cherry trees from the group Sato zakura (Prunus serrulata 'Amanogawa', 'Kanzan' and 'Kiku-shidare-zakura') and two domestic cherry rootstocks -Prunus avium and Prunus 'Colt'. The explants used in the research were: leaf petiole, leaf base with a part of a petiole, part of lamina with a midvein and a stem with an axillary bud. Among three plant growth media (MS, SH and WP) that were used in this study, the MS proved to be the most favourable for the majority of taxa during the callus induction process. For the sweet cherry tree and the cultivars 'Kanzan' and 'Colt', the SH plant growth medium was also acceptable. The best results in callogenesis were obtained for the majority of taxons with auxin at the concentration 2 mgL-1 NAA and cytokinin BAP 0.5 mgL-1. It is also possible to use 2.4-D at the same concentration as a substitute for the genotypes Prunus avium, Prunus ` Colt' and Prunus serrulata 'Kanzan', whereas IBA proved to be an inappropriate auxin for callus induction. The protocol described herein is proved to be efficient callus induction in a range of taxa of genus Prunus