title: Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring.
abstract: Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Royal Institute of Technology (KTH), SE-171 65, Solna, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Kvastad
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Royal Institute of Technology (KTH), SE-171 65, Solna, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Werne Solnestam
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Royal Institute of Technology (KTH), SE-171 65, Solna, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Johansson
authors.affiliation: MRC-Holland, Amsterdam, The Netherlands.|Agena Bioscience, San Diego, California.
authors.forename: A O
authors.initials: AO
authors.lastname: Nygren
authors.affiliation: MRC-Holland, Amsterdam, The Netherlands.
authors.forename: N
authors.initials: N
authors.lastname: Laddach
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Royal Institute of Technology (KTH), SE-171 65, Solna, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Sahlén
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Royal Institute of Technology (KTH), SE-171 65, Solna, Sweden.
authors.forename: S
authors.initials: S
authors.lastname: Vickovic
authors.affiliation: Pathology Dept, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
authors.forename: Schirmer C
authors.initials: SC
authors.lastname: Bendigtsen
authors.affiliation: Pathology Dept, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
authors.forename: M
authors.initials: M
authors.lastname: Aaserud
authors.affiliation: Pathology Dept, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
authors.forename: L
authors.initials: L
authors.lastname: Floer
authors.affiliation: Pathology Dept, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
authors.forename: E
authors.initials: E
authors.lastname: Borgen
authors.affiliation: Fraunhofer ICT-IMM, Mainz, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Schwind
authors.affiliation: Fraunhofer ICT-IMM, Mainz, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Himmelreich
authors.affiliation: Fraunhofer ICT-IMM, Mainz, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Latta
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Royal Institute of Technology (KTH), SE-171 65, Solna, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Lundeberg
mesh_terms: D016022:Case-Control Studies; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D009360:Neoplastic Cells, Circulating; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis / Q000379:methods*