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  • 1.
    Berg, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hedrum, A
    Holmberg, A
    Pontén, F
    Uhlén, M
    Lundeberg, J
    Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides.1995Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 41, nr 10, s. 1461-6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.

  • 2.
    Chinnasamy, Thiruppathiraja
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Segerink, Loes I.
    Nystrand, Mats
    Gantelius, Jesper
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Point-of-Care Vertical Flow Allergen Microarray Assay: Proof of Concept2014Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 60, nr 9, s. 1209-1216Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of < 10 min before imaging and data analysis. METHOD: Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of <= 10 min. Microarray images were captured by a consumer-grade flatbed scanner. RESULTS: A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was < 14%. The observed concordance with a clinical assay, Immuno-CAP, was R-2 = 0.89 (n = 31). CONCLUSIONS: In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.

  • 3.
    Hartmann, Michael
    et al.
    NMI–Natural and Medical Sciences Institute at the University of Tübingen.
    Schrenk, Monika
    Dottinger, Anette
    Nagel, Sarah
    Roeraade, Johan
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Joos, Thomas O.
    Templin, Markus F.
    Expanding assay dynamics: A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays2008Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, nr 6, s. 956-963Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

  • 4. Holmberg, K.
    et al.
    Persson, M. L.
    Uhlén, Mathias
    Odeberg, Jacob
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Pyrosequencing analysis of thrombosis-associated risk markers2005Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 51, nr 8, s. 1549-1552Artikel i tidskrift (Refereegranskat)
  • 5.
    Käller, Max
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Tuominen, Rainer
    Karolinska Institutet.
    Ahmadian, Afshin
    KTH, Skolan för bioteknologi (BIO).
    Magnusson, Veronica
    Karolinska Institutet.
    Egyhazi, Suzanne
    Karolinska Institutet.
    Hansson, Johan
    Karolinska Institutet.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Detection of MC1R Polymorphisms with Protease-Mediated Allele-Specific Extension as an Alternative to Direct Sequencing2005Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 51, nr 12, s. 2388-2391Artikel i tidskrift (Refereegranskat)
  • 6.
    Russom, Aman
    et al.
    Surgical Services and Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston.
    Sethu, Palaniappan
    Irimia, Daniel
    Mindrinos, Michael N.
    Calvan, Steve E.
    Garcia, Iris
    Finnerty, Celeste
    Tannahill, Cynthia
    Abouhamze, Amer
    Wilhelmy, Julie
    Lopez, M. Cecilia
    Baker, Henry V.
    Herndon, David N.
    Lowry, Stephen F.
    Maier, Ronald V.
    Davis, Ronald W.
    Moldawer, Lyle L.
    Tompkins, Ronald G.
    Toner, Mehmet
    Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients2008Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, nr 5, s. 891-900Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. METHODS: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. RESULTS: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. CONCLUSIONS: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting. (c) 2008 American Association for Clinical Chemistry.

  • 7. Sivertsson, A.
    et al.
    Platz, A.
    Hansson, J.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Pyrosequencing as an alternative to single-strand conformation polymorphism analysis for detection of N-ras mutations in human melanoma metastases2002Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 48, nr 12, s. 2164-2170Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Mutations in codons 12, 13, and 61 of the N-ras gene are, common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras. Methods: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively. Results: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis And confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified. Conclusion: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.

  • 8.
    Williams, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Norberg, T
    Ahmadian, A
    Pontén, F
    Bergh, J
    Inganäs, M
    Lundeberg, J
    Uhlén, M
    Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets.1998Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 44, nr 3Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.

1 - 8 av 8
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