Ncounter pancancer progression panel

The remaining cDNAs from the AdnaTest were used for RNA profiling by the nCounter PanCancer Progression Panel (NanoString Technologies, WA). The 770 targeted genes in the panel were amplified using the nCounter Low RNA Input Kit (NanoString Technologies) which has been validated and found to be highly efficient and specific [19 (link)]. Briefly, the cDNAs of CTCs were amplified using the multiplex low-input primer pool with 7 cycles of PCR. The PCR-amplified products were then quantified by an Agilent Bioanalyzer 2100 (Agilent Technologies) and hybridized with the nCounter PanCancer Progression Panel following the standard nCounter hybridization protocol.

The
total RNA of cells and EVs were assayed by the nCounter PanCancer
Progression Panel (NanoString Technologies, WA) to determine the expression
of 770 mRNAs. Since the RNA input amount from EV samples was less
than the minimum requirement for the panel, the targeted genes in
the panel were amplified with a two-step process using the nCounter
Low RNA Input Kit (NanoString Technologies) which has been validated
and found to be highly efficient and specific (Figure 1). Briefly, the EV RNAs were first converted
to cDNA, which were further amplified using the multiplex low-input
primer pool with 14 cycles of PCR. To make the results comparable,
though all cell-derived RNA samples had sufficient RNA for direct
panel analysis, we diluted the cell RNA and used 0.2 ng to go through
the same amplification protocol. The PCR-amplified products were then
quantified by an Agilent Bioanalyzer 2100 (Agilent Technologies) and
hybridized with the nCounter PanCancer Progression Panel following
the standard nCounter hybridization protocol.