Measurement Of Protein Concentration Research Articles

Expression of inflammatory and structural matrix genes in synovial fluid following intra-articular administration of isoflupredone acetate to exercised horses.

Intra-articular use of corticosteroids is commonplace in performance horses. Isoflupredone acetate (IPA) is one of four Food and Drug Administration approved corticosteroids for intra-articular use in horses. The lack of published reports describing the efficacy and duration of effects of this drug warrant further study. To assess the effects of intra-articular administration of IPA on the expression of selected anti- and pro-inflammatory and structural matrix genes following intra-articular administration to exercised Thoroughbred horses and to correlate these effects with drug concentrations. Block design in vivo experiment. Twelve exercised horses received either a single intra-articular administration of 8 mg of IPA or 0.9% saline solution. Synovial fluid samples were collected prior to and up to 42 days post drug administration from the treated joints. Microarray and qRT-PCR analysis were used to assess changes in expression levels of various inflammatory and structural genes post drug administration. On microarray analysis, 855, 23,358 and 26,411 genes had a measurable fold change (increase or decrease in expression levels) when comparing baseline samples to 24 h, baseline samples to day 7 and 24 h samples to day 7, respectively. Of the genes selected for further study by qRT-PCR analysis, expression of ANXA-1 (lipocortin) was significantly increased and IL23A and MMP1 and MMP9 significantly decreased following IPA administration. Expression levels of collagen genes were not significantly different from baseline. Limitations include the use of a noninflammatory model as results may differ in the presence of an acute inflammatory insult and the inability to measure protein concentrations of inflammatory mediators due to limited synovial fluid sample volume. Expression relative to baseline, for both inflammatory and matrix genes for up to 42 days post IPA administration, suggests a prolonged effect relative to detection time in both plasma and synovial fluid.

MHealth Dipstick Analyzer for Monitoring of Pregnancy Complications

Dipstick-based urinalysis is routinely used for the detection of early signs of such pregnancy complications, as preeclampsia and gestational diabetes. Usually it is done in doctor's office using an automatic dipstick analyzer. Here, we present a novel smartphone-based colorimeter and demonstrate its application to the measurements of glucose and protein concentrations in biological samples. The key innovations of our approach was to combine powerful image processing encoded into a mobile phone application with a low cost 3-D printed sample holder that allowed to control lighting conditions and significantly improved sensitivity. Different solutions with protein and glucose concentrations ranging from 0 to 2000 mg/dL were prepared and analyzed using our system. The detection limit for glucose was 40 mg/dL and for protein -6mg/dL The smartphone-based colorimeter always correctly classified the corresponding reagent strip pads, what confirms that it can be used as a low cost alternative for commercial dipstick analyzers.

Developing a NIR multispectral imaging for prediction and visualization of peanut protein content using variable selection algorithms

The feasibility of developing a multispectral imaging method using important wavelengths from hyperspectral images selected by genetic algorithm (GA), successive projection algorithm (SPA) and regression coefficient (RC) methods for modeling and predicting protein content in peanut kernel was investigated for the first time. Partial least squares regression (PLSR) calibration model was established between the spectral data from the selected optimal wavelengths and the reference measured protein content ranged from 23.46% to 28.43%. The RC-PLSR model established using eight key wavelengths (1153, 1567, 1972, 2143, 2288, 2339, 2389 and 2446 nm) showed the best predictive results with the coefficient of determination of prediction (R2P) of 0.901, and root mean square error of prediction (RMSEP) of 0.108 and residual predictive deviation (RPD) of 2.32. Based on the obtained best model and image processing algorithms, the distribution maps of protein content were generated. The overall results of this study indicated that developing a rapid and online multispectral imaging system using the feature wavelengths and PLSR analysis is potential and feasible for determination of the protein content in peanut kernels.

Magnetic Fluorescent Nanoparticles Binding to Amyloid-Beta Peptide: Silica-Coated, Thioflavin-T Functionalized Iron Oxide

Formation and accumulation of extracellular amyloid-beta ( $\text{A}\beta )$ peptide aggregates in the brain are associated with Alzheimer’s disease, a widespread age-related form of dementia, for which there is no effective treatment at present. It is essential to discover ways of visualizing and monitoring the progress of aggregation toward amyloidic plaques while at the same time therapeutic methods inhibiting the aggregation can be explored. Iron oxide nanoparticles Fe3O4 with size ~10 nm have been coated with silica SiO2 and Thioflavin-T (ThT), a non-toxic fluorescent dye binding to the amyloid- $\beta $ structures with a possible effect on fibril formation, and then added to $\text{A}\beta _{\mathrm {1-42}}$ phosphate-buffered saline solution, incubated for 30 min and extracted by magnetic separation. The magnetic ThT silica-coated nanoparticles with size ~20 nm have been characterized structurally and magnetically by powder X-ray powder diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and vibrating sample magnetometer. Their affinity to $\text{A}\beta _{\mathrm {1-42}}$ peptide, examined by fluorescence microscopy and protein concentration measurements using the BCA Protein Assay Kit has been verified. Successful binding of magnetic silica nanoparticles, doped with ThT, to $\text{A}\beta _{\mathrm {1-42}}$ peptide provides a nanocarrier potentially exemplifying multiple functions, such as magnetic imaging, magnetic handling, drug delivery, fluorescence imaging, $\text{A}\beta $ binding, and $\text{A}\beta $ aggregation inhibition.

Optical Sensing of Weed Infestations at Harvest.

Kochia (Kochia scoparia L.), Russian thistle (Salsola tragus L.), and prickly lettuce (Lactuca serriola L.) are economically important weeds infesting dryland wheat (Triticum aestivum L.) production systems in the western United States. Those weeds produce most of their seeds post-harvest. The objectives of this study were to determine the ability of an optical sensor, installed for on-the-go measurement of grain protein concentration, to detect the presence of green plant matter in flowing grain and assess the potential usefulness of this information for mapping weeds at harvest. Spectra of the grain stream were recorded continuously at a rate of 0.33 Hz during harvest of two spring wheat fields of 1.9 and 5.4 ha. All readings were georeferenced using a Global Positioning System (GPS) receiver with 1 m positional accuracy. Chlorophyll of green plant matter was detectable in the red (638–710 nm) waveband. Maps of the chlorophyll signal from both fields showed an overall agreement of 78.1% with reference maps, one constructed prior to harvest and the other at harvest time, both based on visual evaluations of the three green weed species conducted by experts. Information on weed distributions at harvest may be useful for controlling post-harvest using variable rate technology for herbicide applications.

Mathematical model of early Reelin-induced Src family kinase-mediated signaling.

Reelin is a large glycoprotein with a dual role in the mammalian brain. It regulates the positioning and differentiation of postmitotic neurons during brain development and modulates neurotransmission and memory formation in the adult brain. Alterations in the Reelin signaling pathway have been described in different psychiatric disorders. Reelin mainly signals by binding to the lipoprotein receptors Vldlr and ApoER2, which induces tyrosine phosphorylation of the adaptor protein Dab1 mediated by Src family kinases (SFKs). In turn, phosphorylated Dab1 activates downstream signaling cascades, including PI3-kinase-dependent signaling. In this work, a mechanistic model based on ordinary differential equations was built to model early dynamics of the Reelin-mediated signaling cascade. Mechanistic models are frequently used to disentangle the highly complex mechanisms underlying cellular processes and obtain new biological insights. The model was calibrated on time-resolved data and a dose-response measurement of protein concentrations measured in cortical neurons treated with Reelin. It focusses on the interplay between Dab1 and SFKs with a special emphasis on the tyrosine phosphorylation of Dab1, and their role for the regulation of Reelin-induced signaling. Model selection was performed on different model structures and a comprehensive mechanistic model of the early Reelin signaling cascade is provided in this work. It emphasizes the importance of Reelin-induced lipoprotein receptor clustering for SFK-mediated Dab1 trans-phosphorylation and does not require co-receptors to describe the measured data. The model is freely available within the open-source framework Data2Dynamics (www.data2dynamics.org). It can be used to generate predictions that can be validated experimentally, and provides a platform for model extensions both to downstream targets such as transcription factors and interactions with other transmembrane proteins and neuronal signaling pathways.

SI-traceable calibration-free analysis for the active concentration of G2-EPSPS protein using surface plasmon resonance

Active proteins play important roles in the function regulation of human bodies and attract much interest for use in pharmaceuticals and clinical diagnostics. However, the lack of primary methods to analyze active proteins means there is currently no metrology standard for active protein measurement. In recent years, calibration-free concentration analysis (CFCA), which is based on surface plasmon resonance (SPR) technology, has been proposed to determine the active concentration of proteins that have specific binding activity with a binding partner without any higher order standards. The CFCA experiment observes the changes of binding rates at totally different two flow rates and uses the known diffusion coefficient of an analyte to calculate the active concentration of proteins, theoretically required, the binding process have to be under diffusion-limited conditions. Measuring the active concentration of G2-EPSPS protein by CFCA was proposed in this study. This method involves optimization of the regeneration buffer and preparation of chip surfaces for appropriate reaction conditions by immobilizing ligands (G2-EPSPS antibodies) on sensor chips (CM5) via amine coupling. The active concentration of G2-EPSPS was then determined by injection of G2-EPSPS protein samples and running buffer over immobilized and reference chip surfaces at two different flow rates (5 and 100μLmin−1). The active concentration of G2-EPSPS was obtained after analyzing these sensorgrams with the 1:1 model. Using the determined active concentration of G2-EPSPS, the association, dissociation, and equilibrium constants of G2-EPSPS and its antibody were determined to be 2.18 ± 0.03 × 106M−1s−1, 5.79 ± 0.06 ×10−3s−1, and 2.65 ± 0.06 × 10−9M, respectively. The performance of the proposed method was evaluated. The within-day precisions were from 3.26% to 4.59%, and the between-day precision was 8.36%. The recovery rate of the method was from 97.46% to 104.34% in the concentration range of 1.5–8nM. The appropriate concentration range of G2-EPSPS in the proposed method was determined to be 1.5–8nM. The active G2-EPSPS protein concentration determined by our method was only 17.82% of that obtained by isotope dilution mass spectrometry, showing the active protein was only a small part of the total G2-EPSPS protein. The measurement principle of the proposed method can be clearly described by equations and the measurement result can be expressed in SI units. Therefore, the proposed method shows promise to become a primary method for active protein concentration measurement, which can benefit the development of certified reference materials for active proteins.

Suitability of total nitrogen as a predictor of protein content in stream periphyton

AbstractDespite its importance in trophic dynamics, the protein content of food resources is often estimated using N-to-protein conversion factors. This method assumes that all N is contained in proteins and that all proteins are 16% N by mass. However, if the environmental factors affecting protein content differ from those affecting N content, conflating protein and N contents could lead to a misunderstanding of foodweb dynamics. We directly measured the protein content of periphyton collected from 46 streams in southeastern New York State (USA) using a modified Lowry method and compared it to the protein content of the same periphyton estimated with the traditional 6.25 mg protein/mg N conversion factor. We assessed the potential ecological drivers of protein and N contents based on correlations between these 2 metrics and various environmental factors. Directly measured protein content was positively correlated with N content, but mean directly measured protein content was <½ of mean estimated protein...

How well do growing season dynamics of photosynthetic capacity correlate with leaf biochemistry and climate fluctuations?

Accurate values of photosynthetic capacity are needed in Earth System Models to predict gross primary productivity. Seasonal changes in photosynthetic capacity in these models are primarily driven by temperature, but recent work has suggested that photoperiod may be a better predictor of seasonal photosynthetic capacity. Using field-grown kudzu (Pueraria lobata (Willd.) Ohwi), a nitrogen-fixing vine species, we took weekly measurements of photosynthetic capacity, leaf nitrogen, and pigment and photosynthetic protein concentrations and correlated these with temperature, irradiance and photoperiod over the growing season. Photosynthetic capacity was more strongly correlated with photoperiod than with temperature or daily irradiance, while the growing season pattern in photosynthetic capacity was uncoupled from changes in leaf nitrogen, chlorophyll and Rubisco. Daily estimates of the maximum carboxylation rate of Rubisco (Vcmax) based on either photoperiod or temperature were correlated in a non-linear manner, but Vcmax estimates from both approaches that also accounted for diurnal temperature fluctuations were similar, indicating that differences between these models depend on the relevant time step. We advocate for considering photoperiod, and not just temperature, when estimating photosynthetic capacity across the year, particularly as climate change alters temperatures but not photoperiod. We also caution that the use of leaf biochemical traits as proxies for estimating photosynthetic capacity may be unreliable when the underlying relationships between proxy leaf traits and photosynthetic capacity are established outside of a seasonal framework.

A Nondestructive Method for Measuring Protein Distribution in Frozen Drug Substance

We describe a noninvasive method developed to make in situ measurements of protein concentration in frozen drug substance. This technique is based on fluorescence from artificially labeled protein and a charge-coupled device camera. Data collected using this method in laboratory small-scale experiments are in good agreement with traditional ice core method. The technique allows real-time visualization of freezing process and provides rich local details of ice crystal growth and morphology for the whole freezing process from beginning to the last point to freeze, and the whole freezing process can be described in 2- and 3-dimensional heat maps with appropriate software. In combining with other existing methods, this method can provide evaluation and optimization of formulation, cooling rate, and cryoconcentration distribution and impacts of combined stresses during freezing. The ability to understand and to control the protein concentration profile in the frozen state offers the potential to improve stability of protein in long-term frozen storage.

Allergenic extracts to diagnose and treat sensitivity to insect venoms and inhaled allergens

ObjectiveTo review allergenic extracts used to diagnose or treat insect allergies, including how the extracts are manufactured and their measurements of potency or concentration. Data SourcesPeer-reviewed articles derived from searching PubMed (National Center for Biotechnology Information) about insect allergies and extract preparation. Encyclopedia of Life (http://www.eol.org/) and http://allergome.org/ were also referenced for background information on insects and associated allergens. Study SelectionsSearch terms used for the PubMed searches included insect allergens and allergies, Apidae, Vespidae, fire ants, cockroach allergies, insect allergen extract preparation, and standardization. ResultsHumans may be sensitized to insect allergens by inhalation or through stings. Cockroaches and moths are predominantly responsible for inhalation insect allergy and are a major indoor allergen in urban settings. Bees, fire ants, and wasps are responsible for sting allergy. In the United States, there are multiple insect allergen products commercially available that are regulated by the US Food and Drug Administration. Of those extracts, honeybee venom and insect venom proteins are standardized with measurements of potency. The remaining insect allergen extracts are nonstandardized products that do not have potency measurements. ConclusionSensitization to inhalational and stinging insect allergens is reported worldwide. Crude insect allergen extracts are used for diagnosis and specific immunotherapy. A variety of source materials are used by different manufacturers to prepare these extracts, which may result in qualitative differences that are not reflected in measurements of potency or protein concentration.

A Stepwise Integrated Approach to Personalized Risk Predictions in Stage III Colorectal Cancer.

Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a stand-alone signature and as a constituent of further refined prognostic stratification tools.Experimental Design: Apoptosis competency of primary tumor samples from patients with stage III colorectal cancer (n = 120) was calculated by APOPTO-CELL from measured protein concentrations of Procaspase-3, Procaspase-9, SMAC, and XIAP. An enriched APOPTO-CELL signature (APOPTO-CELL-PC3) was synthesized to capture apoptosome-independent effects of Caspase-3. Furthermore, a machine learning Random Forest approach was applied to APOPTO-CELL-PC3 and available molecular and clinicopathologic data to identify a further enhanced signature. Association of the signature with prognosis was evaluated in an independent colon adenocarcinoma cohort (TCGA COAD, n = 136).Results: We identified 3 prognostic biomarkers (P = 0.04, P = 0.006, and P = 0.0004 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively) with increasing stratification accuracy for patients with stage III colorectal cancer.The APOPTO-CELL-PC3 signature ranked highest among all features. The prognostic value of the signatures was independently validated in stage III TCGA COAD patients (P = 0.01, P = 0.04, and P = 0.02 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively). The signatures provided further stratification for patients with CMS1-3 molecular subtype.Conclusions: The integration of a systems-biology-based biomarker for apoptosis competency with machine learning approaches is an appealing and innovative strategy toward refined patient stratification. The prognostic value of apoptosis competency is independent of other available clinicopathologic and molecular factors, with tangible potential of being introduced in the clinical management of patients with stage III colorectal cancer. Clin Cancer Res; 23(5); 1200-12. ©2016 AACR.

A photothermal biosensor for detection of C-reactive protein in human saliva

In this study, our objective was to develop a photothermal biosensor (PTB) for measurement of C-reactive protein (CRP) concentrations in human saliva. The resulting PTB that we developed is rapid, simple, and minimally invasive, and can be used to measure the concentration of CRP in human saliva (7-μL sample volume). The CRP (0.1–100ng/mL) is captured by the sandwich immunoassay on the sensor, and gold nanoparticles (GNPs) conjugated with antibodies cause a photothermal effect when irradiated with a laser (90s, wavelength=532nm). The temperature changes caused by this effect correlate with the concentrations of CRP. Therefore, the sensor shows a higher correlation with the concentrations of CRP than commercial detection kits. Its detection limit is 0.1ng/mL CRP.

New applications of near‐infrared spectroscopy in crop and soil analysis

New applications of near‐infrared spectroscopy in crop and soil analysis

Isolation of Oat (Avena sativa L.) Total Proteins and Their Prolamin Fractions for 2D Electrophoresis.

Appropriate sample preparation is essential to obtaining good results of two-dimensional gel electrophoresis (2-DE). For various reasons (particularly phenolic compounds, proteolytic enzymes, and cell-wall mucilages) the extraction of proteins from plant material, among them oat proteins, is difficult. During isolation all soluble substances that may interfere with the analysis (especially isoelectric focusing) are removed, and proteins of interest are separated from the remains. However, the applied procedure of isolation cannot be too extensive, because additional stages cause loss of the proteins.In this chapter, we describe a simple procedure for the isolation of oat total proteins and their prolamin fractions prior to 2-DE, without necessity of considerable purification. It can be used for oat protein fractionation, measurement of oat protein concentration, and their 2-DE analysis, with particular reference to prolamin fractions. The presented routine includes modified methods of plant seed proteins extraction and sequential Osborne extraction, based on oat protein solubility differences.

Quantitative Proteomics Using SILAC.

The ability to enumerate all of the proteins in a cell is quickly becoming a reality. Quantitative proteomics adds an extra dimension to proteome-wide discovery experiments by enabling differential measurements of protein concentrations, characterization of protein turnover, increased stringency of co-immunoprecipitation reactions, as well as many other intriguing applications. One of the most widely used techniques that enable relative protein quantitation is stable isotope labeling by amino acids in cell culture (SILAC) (Ong et al., Mol Cell Proteomics 1(5):376-386, 2002). Over the past decade, SILAC has become the preferred approach for proteome-wide quantitation by mass spectrometry. This approach relies on the metabolic incorporation of isotopically enriched amino acids into the proteome of cells-the proteome of "light" (1H, 12C, 14N) cells can then be compared to "heavy" (2H, 13C, 15N) cells as the isotopically labeled proteins and peptides are easily distinguished in a mass spectrometer. Since cellular uptake and response to isotopically different amino acid(s) is naïve, it is without impact on cell physiology. We provide a detailed step-by-step procedure for performing SILAC-based experiment for proteome-wide quantitation in this chapter.

A Rapid Method for Measuring Seed Protein Content in Cowpea (&amp;lt;i&amp;gt;Vigna unguiculata&amp;lt;/i&amp;gt; (L.) Walp)

Cowpea (Vigna unguiculata (L.) Walp), a legume crop that is grown in the worldwide, provides beneficial proteins for human consumption and animal feeding. In comparison, Rapid N analyzer as traditional method, has been widely used to measure protein content through the percentage of total nitrogen in the seed’s grounded powder. Near-Infrared Reflectance (NIR) has commonly been used to measure protein content in soybean seeds using whole grain without the need of seed grinding, which makes it possible to obtain fast results at a lower cost-per-analysis than the traditional combustion method. The specific objective of this study is to test a rapid method for measuring cowpea seed protein content by the NIR analyzer comparing to the traditional rapid N analyzer. A total of 240 cowpea genotypes were used in this study, including six seed coat colors, black, blackeye, browneye, cream, pinkeye, and red with 40 cowpea genotypes. The results showed that a linear relationship exists between the NIR analyzer and the Rapid N analyzer in the six different color groups. The correlation efficiency (r) between the seed protein contents from NIR and Rapid N was higher for pinkeye seed (r = 0.867), blackeye (0.771), cream (0.729), browneye (0.700), and red (0.623), respectively, but lower for black seeds, indicating that the NIR analyzer can be used to measure protein content for cowpea seeds with the five types of seed coat except black. Overview, the cowpea seed protein content measured from the NIR analyzer showed a little higher seed protein content. A series of regression models with different seed coat color have been built to adjust to protein content of colorful cowpea seeds from the NIR analyzer. But, it is not recommended to use for black color seeds due to a very low correlation efficiency (r) value with 0.184.

Impact of feeding and housing systems on disease incidence in dairy calves

Contentious issues in calf rearing include milk feeding level and single versus group housing. The current study was performed on a high-producing 170 Holstein cow dairy farm to investigate the...

Measurement of serum C-reactive protein concentration for discriminating between suppurative arthritis and osteoarthritis in dogs.

BackgroundIn a dog with joint pain, it is important to determine whether it has suppurative joint disease, characterized by exudation of neutrophils in the synovial fluid, or not, as this affects choice of diagnostic tests and treatments. The aim of this study was to evaluate whether measurement of serum C-reactive protein (CRP) concentration could be used to discriminate between dogs with suppurative arthritis and osteoarthritis (OA). Furthermore, the concentrations of serum and synovial fluid interleukin (IL) 6 concentrations were measured in dogs with joint disease and in healthy dogs, and were correlated to serum CRP concentrations.MethodsDogs with joint pain were enrolled prospectively and were classified to have suppurative arthritis or OA based on synovial fluid analysis and radiographic/arthroscopic findings. Healthy Beagles were enrolled as a comparative group. CRP and IL-6 concentrations were measured with canine-specific immunoassays. The performance of CRP concentration in discriminating between dogs with suppurative arthritis and OA was evaluated using a previously established clinical decision limit for CRP (20 mg/l), and by receiver operator characteristic (ROC) curve and logistic regression analysis. Comparisons of CRP and IL-6 concentrations between groups were performed using t-tests, and correlations by Spearman rank correlation coefficients.ResultsSamples were obtained from 31 dogs with suppurative arthritis, 34 dogs with OA, and 17 healthy dogs. Sixty-two out of 65 dogs with joint disease were correctly classified using the clinical decision limit for CRP. Evaluation of ROC curve and regression analysis indicated that serum CRP concentrations could discriminate between suppurative arthritis and OA. Dogs with suppurative arthritis had higher serum CRP and serum and synovial fluid IL-6 concentrations compared to dogs with OA (p < 0.001). Dogs with OA had higher synovial fluid IL-6 concentrations (p < 0.001), but not higher serum CRP (p = 0.29) or serum IL-6 (p = 0.07) concentrations, compared to healthy dogs. There was a positive correlation between synovial fluid IL-6 and serum CRP concentrations (rs = 0.733, p < 0.001), and between serum IL-6 and serum CRP concentrations (rs = 0.729, p < 0.001).ConclusionCRP concentration was found to discriminate well between dogs with suppurative arthritis and OA.

Development of Marker-Free Insect-Resistant Indica Rice by Agrobacterium tumefaciens-Mediated Co-transformation.

Agrobacterium-mediated co-transformation is an efficient strategy to generate marker-free transgenic plants. In this study, the vectors pMF-2A∗ containing a synthetic cry2A∗ gene driven by maize ubiquitin promoter and pCAMBIA1301 harboring hygromycin phosphotransferase gene (hpt) were introduced into Minghui86 (Oryza sativa L. ssp. indica), an elite indica restorer line. Two independent transformants containing both the cry2A∗ gene and hpt gene were regenerated. Several homozygous marker-free transgenic progenies were derived from family 2AH2, and three of them were selected for further insect bioassay in the laboratory and field. Insect-resistance assays revealed that all the three transgenic lines were highly resistant to striped stem borer (Chilo suppressalis), yellow stem borer (Tryporyza incertulas) and rice leaf folder (Cnaphalocrocis medinalis). The measurement of Cry2A protein concentration showed that Cry2A protein was stably expressed in leaves and stems of homozygous transgenic lines and their hybrids. The yields of the marker-free homozygous transgenic lines and their hybrids were not significantly different from those of their corresponding controls. Furthermore, the results of flanking sequence isolation showed that the T-DNA in line 8-30 was integrated into the intergenic region of chromosome 2 (between Os02g43680 and Os02g43690). These results indicate that the marker-free transgenic rice has the potential for commercial production.