| Autor: |
Acero Sánchez JL; Universitat Rovira i Virgili , Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain., Joda H; Universitat Rovira i Virgili , Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain., Henry OY; Universitat Rovira i Virgili , Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain., Solnestam BW; KTH Royal Institute of Technology , Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden., Kvastad L; KTH Royal Institute of Technology , Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden., Akan PS; KTH Royal Institute of Technology , Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden., Lundeberg J; KTH Royal Institute of Technology , Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65 Solna, Sweden., Laddach N; MRC-Holland , Willem Schoutenstraat 1, 1057 DL Amsterdam, The Netherlands., Ramakrishnan D; Labman Automation Ltd. , Seamer Hill, Seamer, Stokesley, North Yorkshire TS9 5NQ, United Kingdom., Riley I; Labman Automation Ltd. , Seamer Hill, Seamer, Stokesley, North Yorkshire TS9 5NQ, United Kingdom., Schwind C; Fraunhofer (ICT-IMM) , Carl-Zeiss-Str. 18-20, 55129 Mainz, Germany., Latta D; Fraunhofer (ICT-IMM) , Carl-Zeiss-Str. 18-20, 55129 Mainz, Germany., O'Sullivan CK; Universitat Rovira i Virgili , Departament de Enginyeria Química, Av. Països Catalans 26, 43007 Tarragona, Spain.; Institució Catalana de Recerca i Estudis Avançats , Passeig Lluís Companys 23, 08010 Barcelona, Spain. |
| Abstrakt: |
Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity. |
