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Results for “protocol optimization”

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PubMed2004

Profiling gene expression in whole blood samples following an in-vitro challenge.

Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.

Adult↗

PubMed2004

Optimization and application of the group I ribozyme trans-splicing reaction.

Group I ribozymes are naturally occurring catalytic RNAs that are able to excise themselves as introns (group I introns) from a precursor RNA, and to ligate the flanking exons. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence specific manner, and to covalently link an RNA sequence to this separate RNA molecule. This ribozyme transesterification reaction has potential in molecular biology and in medicine as a new approach to gene therapy. Here we describe detailed optimized protocols where trans-splicing group I ribozymes are applied in mapping accessible sites in target messenger RNA, and in messenger RNA-repair by correcting mutations.

Animals↗

PubMed2004

Two-dimensional gel electrophoresis for the identification of signaling targets.

Two-dimensional electrophoresis (2-DE) is a powerful technique to differentially display patterns of protein expression and posttranslational modifications, providing a good strategy to monitor molecular responses induced by the activation or inactivation of specific signaling pathways. In this chapter, optimized protocols for 2-DE using extracts from tissue culture are provided. Protocols for in-gel digestion of gel-resolved proteins, which allow protein identification by mass spectrometry are also discussed.

Cell Line, Tumor↗

PubMed2005

RNA synthesis using 2'-O-(tert-butyldimethylsilyl) protection.

This chapter enables the reader to carry out the solid-phase synthesis of ribonucleic acid (RNA) using beta-cyanoethyl phosphoramidite chemistry combined with tert-butyldimethylsilyl protection of the ribose 2'-hydroxyl group. Phosphoramidite monomers are activated with 5-benzylmercapto-1H-tetrazole enabling fast and highly efficient coupling to the 5'-hydroxyl group of the support-bound oligonucleotide. On completion of the synthesis, the stepwise deprotection of the nucleobase, phosphate, and ribose protecting groups is carried out using optimized protocols. Subsequently the various high-pressure (performance) liquid chromatography (HPLC) procedures are described enabling the purification and analysis of the RNA. For this purpose anion-exchange and reversed-phase HPLC are used singly or in combination according to the final purity requirement of the RNA.

Chromatography, High Pressure Liquid↗

PubMed2005

Electroporation: introduction and expression of transgenes in plant protoplasts.

An optimized protocol for the electroporation-based transfection of tobacco protoplasts is described that routinely results in transgene expression frequencies approaching 90%. The overall efficiency of the procedure depends collectively on numerous key parameters, including protoplast viability; DNA concentration, purity, and topology; carrier DNA; and electrical conditions such as ionic strength of the electroporation buffer, electric field strength, pulse duration, and capacitance. Individual methodologies that address each one of these parameters are presented in sufficient detail to enable successful reproduction of this method along with notes that describe helpful tips.

Electroporation↗

PubMed2005

Laser-assisted microdissection of membrane-mounted sections following immunohistochemistry and in situ hybridization.

Laser microbeam microdissection (LMM) is an increasingly important histological technique for obtaining homogeneous cell populations and tissue components in order to analyze target-specific changes in genes, gene expression, and proteins. The quality of data obtained with LMM is heavily dependent on the precision with which the target for microdissection can be identified. Since no cover slip is used during LMM, tissue morphology is poor compared with traditional light microscopy. This hampers morphological recognition of targets for microdissection in routinely stained sections and can be a limiting factor in the use of this technique. Immunohistochemistry (IHC) and in situ hybridization (ISH) can improve the identification of specific cell populations in situ in tissue sections, but there are a number of problems in applying these methods to slides prepared for LMM. In this chapter, we present optimized protocols that allow IHC to be performed for detecting a wide range of antigens in conjunction with LMM, both on formalin-fixed paraffin-embedded and on frozen sections. In addition, we present a quick, versatile protocol for performing ISH on archival material suitable for LMM.

Animals↗

PubMed2005

Magnetic resonance imaging of amyloid plaques in transgenic mice.

Transgenic mice are used increasingly to model brain amyloidosis, mimicking the pathogenic processes involved in Alzheimer's disease (AD). In this chapter, a strategy is described that has been successfully used to map amyloid deposits in transgenic mouse models of AD with magnetic resonance imaging (MRI), utilizing molecular targeting vectors labeled with MRI contrast agents to enhance selectively the signal from amyloid plaques. To obtain sufficient spatial resolution for effective and sensitive mouse brain imaging, magnetic fields of 7-Tesla (T) or more are required. These are higher than the 1.5-T field strength routinely used for human brain imaging. The higher magnetic fields affect contrast agent efficiency, and determine the choice of pulse sequence parameters for in vivo MRI, all addressed in this chapter. Ex vivo imaging is also described as an important step to test and optimize protocols prior to in vivo studies. The experimental setup required for mouse brain imaging is explained in detail, including anesthesia, immobilization of the mouse head to reduce motion artifacts, and anatomical landmarks to use for the slice alignment procedure to improve image co-registration during longitudinal studies, and for subsequent matching of MRI with histology.

Amyloid beta-Peptides↗

PubMed2007

Agrobacterium-mediated transformation to create an insertion library in Magnaporthe grisea.

Magnaporthe grisea is the causal agent of rice blast disease and represents a model organism for the study of fungal plant-pathogen interactions. Pathogenicity is a complex phenotype, which is carefully orchestrated by the fungus and begins with recognition and infection of the host plant, followed by growth within the plant cells, and finally dissemination to the next host and continuation of the fungal life cycle. Certain genes must condition the ability of a pathogenic fungus to infect and cause disease symptoms. To learn more about the infection process and the genes that are involved in the complex interplay between M. grisea and rice, we used an insertional mutagenesis approach to attempt to randomly disrupt all genes in the fungal genome. Two transformation approaches were used to build a library of insertion strains in M. grisea. Polyethylene glycol/CaCl2-mediated protoplast transformation was the initial method we used and resulted in the generation of just more than 17,000 insertion strain lines. Later Agrobacterium tumefaciens-mediated transformation was adopted and the final number of insertional mutant strains of M. grisea strain 70-15 generated was more than 57,000. Here, we describe the methods used for A. tumefaciens-mediated transformation of M. grisea and the optimized protocols we have developed to enable high-throughput fungal transformation.

Agrobacterium tumefaciens↗

PubMed2006

Generation and characterization of monkey embryonic stem cells.

Nonhuman primate embryonic stem (ES) cells are very important for preclinical research of the medical application of human ES cells. Because primate ES cells show significant differences from mouse ES cells, we have been optimizing protocols for the establishment and maintenance of monkey ES cells. The latest methods for derivation and culture of cynomolgus monkey ES cells are described in detail.

Animals↗

PubMed2006

Genomic sequencing of the severe acute respiratory syndrome-coronavirus.

The polymerase chain reaction (PCR), which can exponentially replicate a target DNA sequence, has formed the basis for the sensitive and direct examination of clinical samples for evidence of infection. During the epidemic of severe acute respiratory syndrome (SARS) in 2003, PCR not only offered a rapid way to diagnose SARS-coronavirus (SARS-CoV) infection, but also made the molecular analysis of its genomic sequence possible. Sequence variations were observed in the SAR-CoV obtained from different patients in this epidemic. These unique viral genetic signatures can be applied as a powerful molecular tool in tracing the route of transmission and in studying the genome evolution of SARS-CoV. To extract this wealth of information from the limited primary clinical specimens of SARS patients, we were presented with the challenge of efficiently amplifying fragments of the SARS-CoV genome for analysis. In this chapter, we will discuss how we managed to accomplish this task with our optimized protocols on reverse-transcription, nested PCR amplification, and DNA cycle sequencing. We will also discuss the sequence variations that typified some strains of SARS-CoV in the different phases during this epidemic. PCR amplification of the viral sequence and genomic sequencing of these critical sequence variations of re-emerging SARS-CoV strains would give us quick insights into the virus.

DNA Primers↗

PubMedDate not supplied

New homologous bioassays for human lactogens show that agonism or antagonism of various analogs is a function of assay sensitivity.

The reference bioassay for lactogens is the Nb2 cell proliferation assay, whose extreme sensitivity allows the detection of very low amounts of lactogenic activity in biologic fluids. The use of rat Nb2 cells raises the problem of species specificity when analyzing lactogens of other origin, including human lactogenic hormones for which no reference bioassay currently exists. In this article, we describe two new homologous bioassays for human lactogens. One is a transcriptional bioassay generated by stably transfecting 293 human embryonic kidney fibroblasts using two plasmids, encoding the human prolactin receptor (hPRLR) and the PRL-responsive lactogenic hormone response element luciferase reporter gene. The second is a proliferation assay obtained by stably transfecting Ba/F3 cells with a plasmid encoding the hPRLR. We provide characterization of the various clones or cell populations that were isolated, and we describe experiments that were performed to achieve optimized protocols for both bioassays. These new assays were compared with other cells types exhibiting well-recognized PRL-mediated responses (proliferation of Nb2 or of human breast tumor cell lines), using various lactogen analogs. This comparative analysis provides strong evidence that the intrinsic characteristics of each bioassay dramatically affect the biologic properties attributed to the lactogen of interest. Depending on the assay, a given analog can exhibit agonistic or antagonistic properties. We hypothesize that in addition to species specificity, assay sensitivity is the key parameter in directing the apparent bioactivity of lactogens. Of course, in the end, it will be necessary to confirm the agonistic or antagonistic properties of the tested analogs, in vivo.

Animals↗

PubMed2003

Bone mineral density in adult patients with Turner's syndrome: analyses of the effectiveness of GH and ovarian steroid hormone replacement therapies.

To analyze the effects of treatments with GH and cyclic estrogen/progesterone (E/P) replacement on bone mineralization in patients with Turner's syndrome (TS), bone mineral density (BMD) was measured longitudinally. BMDs of the whole body and the lumbar spine in 16 adult female patients with TS (17-38 year old; 0-20 years by length of E/P treatment) were assessed using dual energy X-ray absorptiometry one to 5 times over a treatment period of up to 7 years maximum. GH treatment was performed in 9 cases (GH group), but not in the remaining 7 (non-GH group). E/P replacement therapy was initiated in all patients after they finished GH administration. The BMDs of both the whole body and the lumbar spine in the patients with TS were significantly less than those in age-matched normal subjects, and did not improve with E/P treatment. Although there were no differences in final body height and age at the beginning of E/P administration between the GH and non-GH groups, whole body BMD in the GH group was significantly lower than that in the non-GH group. These results indicate that GH administration in childhood and adolescence and E/P treatment in adulthood did not increase bone mineralization in the TS patients. Therefore, we can conclude that the optimal protocol of hormonal replacement therapy with GH and E/P during childhood and adolescence should be established as soon as possible.

Absorptiometry, Photon↗

PubMed2001

Short-term storage of cane toad (Bufo marinus) gametes.

The responses of cane toad (Bufo marinus) gametes, used as a model for the development of assisted reproduction techniques for rare and endangered amphibians, to short-term storage at temperatures > 0 degrees C were studied. Whole excised testes were stored at 0 degrees or 4 degrees C for 15 days, and sperm motility was measured at excision and after storage for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed > 50% motility for 7 days at 0 degrees C and for 5 days at 4 degrees C. At 15 days, only spermatozoa stored at 0 degrees C still showed some motility (3%). Sperm suspensions were prepared at 5 day intervals over 30 days in simplified amphibian ringer (SAR) at dilutions of 1:1, 1:5 and 1:10 (w/v) testes:SAR. Aliquots from each dilution were stored at 0 degrees C in Eppendorf tubes opened at 5 day intervals of storage (aerated) or kept sealed (unaerated) (treatments: aerated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). After 30 days, sperm motility and fertilizing capacity were determined. The optimal protocol for sperm storage up to 10 days, as assessed by the retention of fertilizing capacity, was as a 1:5 testis:SAR (w/v) suspension, whereas the longest absolute retention of both motility and fertilizing capacity was observed in concentrated (1:1 dilution), anaerobic suspensions (up to 25-30 days). Oviductal oocytes placed in SAR at 5, 10, 15, 20 and 25 degrees C immediately after ovulation lost viability when cooled rapidly to 5 degrees C and stored for 2 h. However, oocytes retained viability for up to 8 h at the optimum storage temperature of 15 degrees C. Thus, it is concluded that during short-term storage spermatozoa retain viability for longer than oocytes, and that spermatozoa in suspensions retain viability for longer than spermatozoa stored in situ in excised testes.

Animals↗

PubMed2004

Observations from multidetector CT imaging of different types of renal artery stents.

PURPOSE: To present an optimized protocol for acquisition and reconstruction of multidetector computed tomographic angiographic (CTA) images of the stents most commonly used in renal arteries. TECHNIQUE: CTA was performed on a 16-detector row CT scanner using 0.75-mm collimation. Multiplanar reformatted images perpendicular to the stents and 2-dimensional curved reformatted images were displayed. Two different view windows ("vascular" and "stent") were used, each adapted to the stent density, the vascular wall density, and the aortic enhancement. Five different types of stainless steel balloon-expandable stents were examined; all caused discernable artifacts. These artifacts became more prominent as the stent density increased, becoming most significant when 2 stents were positioned one inside the other. The "stent" window allowed better appreciation of the stent shape and its position compared to the aortic wall and ostial calcifications. The "vascular" window afforded a better view of the vascular lumen, in addition to visualizing the stent in several planes. CONCLUSIONS: Multidetector CTA using dedicated acquisition and reconstruction protocols is capable of visualizing the vascular lumen of different types of renal stents while avoiding metallic artifacts.

Angiography↗

PubMed2006

Glioma-specific antigens for immune tumor therapy.

This review describes glioma-specific antigens important in immunotherapy of glioma tumors. The structure and function of these antigens and recent immunotherapy data are summarized. Also, some important aspects of tumor formation are outlined. The roles of neuronal precursor cells and tumor stroma cells are discussed. The stroma cells of the tumor may be of interest as a target for tumor therapy, especially since they are less heterogeneous than the tumor cells. To date, the clinical benefit of immunotherapy has been very limited. Immunotherapy is, however, still an extremely promising approach to tumor therapy and it will most likely be implemented as a future treatment option for many types of tumors. The current shortcomings of immunotherapy will probably diminish as we start to understand and are able to modulate tumor-induced immunosuppression. There is also a need for a continued search for new tumor-specific antigens and to optimize protocols for vaccine administration.

Antigens, Neoplasm↗

PubMed2004

[Patients with ischemic stroke are taken care quickly in São Paulo Hospital].

We observed inpatients with the diagnosis of ischemic stroke hospitalized at least 24 hours. We investigated the reasons for early or late arrival to the Emergency Room, the time span from arrival to computed tomography (CT) scanning, factors associated to hospitalization time, and patients outcome. We concluded that people who are cared for by São Paulo Hospital now get to the hospital sooner than they did 3 years ago (47% arrived in 3 hours or less) and that the patient flow inside the hospital became much faster, once any stroke patient is considered now a medical emergency by the hospital staff. Infections were the main complications presented by our patients, which is leading us to optimize protocols to improve emergency care by all the staff.

Acute Disease↗

PubMed2001

Nestin-specific green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for transplantation.

Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.

Animals↗

PubMed2003

Advances in the use of dendritic cells and new adjuvants for the development of therapeutic vaccines.

The recent advances in immunology and biotechnology have opened new perspectives for the development of immunotherapy strategies against cancer and infectious diseases. The understanding of the pivotal role of dendritic cells in the initiation and regulation of the immune response has led to an ensemble of preclinical studies and pilot clinical trials, which have provided some evidence on the potential advantages of using dendritic cells as cellular adjuvants for the development of therapeutic vaccines against infectious diseases and malignancies. Current research efforts are focused on the definition of optimal protocols for dendritic cell-based therapies in patients. An additional area of emerging importance in the field of immunotherapy is the identification of safe, selective, and more powerful adjuvants, capable not only of enhancing immune protection against pathogens, but also of breaking tolerance against certain tumor-associated antigens, which is the critical issue for the development of cancer vaccines. The recent recognition of the key role of certain cytokines, such as type I interferons, in linking the innate and adaptive immunity through their action on dendritic cells opens new perspectives for using these natural factors as adjuvants for the development of therapeutic vaccines. We review some of the emerging research aspects in immunotherapy, with special attention to the perspectives of using new adjuvants and dendritic cell-based vaccines for the treatment of cancer and infectious diseases.

Animals↗