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1.
Sci Rep ; 10(1): 15251, 2020 09 17.
Article in English | MEDLINE | ID: mdl-32943655

ABSTRACT

Single cell transcriptomics is revolutionising our understanding of tissue and disease heterogeneity, yet cell type identification remains a partially manual task. Published algorithms for automatic cell annotation are limited to known cell types and fail to capture novel populations, especially cancer cells. We developed northstar, a computational approach to classify thousands of cells based on published data within seconds while simultaneously identifying and highlighting new cell states such as malignancies. We tested northstar on data from glioblastoma, melanoma, and seven different healthy tissues and obtained high accuracy and robustness. We collected eleven pancreatic tumors and identified three shared and five private neoplastic cell populations, offering insight into the origins of neuroendocrine and exocrine tumors. Northstar is a useful tool to assign known and novel cell type and states in the age of cell atlases.


Subject(s)
Algorithms , Glioblastoma/classification , Glioblastoma/pathology , Melanoma/classification , Melanoma/pathology , Brain/cytology , Cluster Analysis , Databases, Factual , Gene Expression Profiling , Humans , Neuroendocrine Tumors/classification , Neuroendocrine Tumors/pathology , Pancreas, Exocrine/pathology , Pancreatic Neoplasms/classification , Pancreatic Neoplasms/pathology , Single-Cell Analysis
3.
PLoS One ; 8(7): e69813, 2013.
Article in English | MEDLINE | ID: mdl-23874999

ABSTRACT

Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.


Subject(s)
Biological Assay/methods , Real-Time Polymerase Chain Reaction/methods
4.
Nat Protoc ; 8(6): 1234-48, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23722261

ABSTRACT

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.


Subject(s)
Antibodies/metabolism , Immunoassay/methods , Proteins/analysis , Proteins/isolation & purification , Oligonucleotides/genetics , Real-Time Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods
5.
PLoS One ; 7(7): e40405, 2012.
Article in English | MEDLINE | ID: mdl-22808155

ABSTRACT

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.


Subject(s)
Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Protein Interaction Mapping/methods , Proteins/metabolism , Animals , Cell Extracts , Cell Line , Humans , Protein Binding
6.
PLoS One ; 7(2): e31068, 2012.
Article in English | MEDLINE | ID: mdl-22383994

ABSTRACT

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.


Subject(s)
Bioterrorism , Nucleic Acid Amplification Techniques , Bacillus/genetics , Bacillus/metabolism , DNA/genetics , DNA Ligases/metabolism , DNA, Circular/analysis , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescent Dyes/pharmacology , Microscopy, Confocal/methods , Models, Genetic , Oligonucleotide Probes , Oligonucleotides/genetics , Pantoea/genetics , Pantoea/metabolism
7.
Expert Rev Proteomics ; 9(1): 21-32, 2012.
Article in English | MEDLINE | ID: mdl-22292821

ABSTRACT

Improved protein assays promise to offer new insights into biological processes as well as the identification of new, clinically important biomarkers. In recent years, a number of approaches have been developed where protein-binding reagents, typically antibodies, are equipped with DNA strands to enable protein analyses via powerful nucleic acid detection reactions for improved performance. In this review, we provide a background to this emerging field, and we describe several different ways in which these reagents can improve protein analyses by lowering detection thresholds, improving multiplexing and extending the range of biomolecules available for analysis, both in research settings and in clinical routine.


Subject(s)
DNA/chemistry , Proteins/analysis , Biomarkers , Polymerase Chain Reaction
8.
Anal Chem ; 84(4): 1824-30, 2012 Feb 21.
Article in English | MEDLINE | ID: mdl-22248085

ABSTRACT

Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. Although discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed.


Subject(s)
Biomarkers/analysis , Biomarkers/blood , Blood Proteins/analysis , Proteome/analysis , Proteomics/methods , Humans
9.
PLoS One ; 6(9): e25583, 2011.
Article in English | MEDLINE | ID: mdl-21980495

ABSTRACT

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.


Subject(s)
Immunoassay/methods , Proteomics/methods , Sequence Analysis, DNA/methods , Biomarkers/blood , Blood Proteins/analysis , Blood Proteins/genetics , Humans , Immunoassay/economics , Multivariate Analysis , Proteomics/economics , Sequence Analysis, DNA/economics , Time Factors
10.
Mol Cell Proteomics ; 9(2): 327-35, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19955079

ABSTRACT

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.


Subject(s)
Blood Proteins/analysis , Immunoassay/methods , Microspheres , Enzyme-Linked Immunosorbent Assay , Growth Differentiation Factor 15/blood , Humans
11.
Nucleic Acids Res ; 36(8): e45, 2008 May.
Article in English | MEDLINE | ID: mdl-18346972

ABSTRACT

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.


Subject(s)
Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis/methods , Proteins/analysis , RNA, Messenger/analysis , Actins/genetics , Actins/metabolism , Cell Line , Molecular Probes , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/analysis
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