posted on 2017-02-17, 00:00authored byJosep Ll. Acero Sánchez, Hamdi Joda, Olivier Y. F. Henry, Beata W. Solnestam, Linda Kvastad, Pelin S. Akan, Joakim Lundeberg, Nadja Laddach, Dheeraj Ramakrishnan, Ian Riley, Carmen Schwind, Daniel Latta, Ciara K. O’Sullivan
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.