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. 2021 Feb 5;11:598118. doi: 10.3389/fgene.2020.598118

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Copyright © 2021 van de Stolpe, Verhaegh, Blay, Ma, Pauwels, Pegram, Prenen, De Ruysscher, Saba, Slovin, Willard-Gallo and Husain.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Measurement of activity of multiple signaling pathways simultaneously in a single small tissue sample, using RNA pre-amplification by multiplex PCR (96 primer sets) (Philips MPDx/OncoSignal, Eindhoven, Netherlands). Measurement of activity of the MAPK-AP1 (AP1), AR, ER, PI3K-FOXO (PI3K pathway activity is the inverse of measured FOXO activity) pathways in two independent formalin fixed paraffin embedded (FFPE) samples, activity score 0–100 on the y-axis. Left: direct high = pathway activity measurement without pre-amplification in a sufficiently large sample (n = 8), 1 ng per reaction, average mRNA qPCR Cq of 30. Middle: preamp = measurement in a very low RNA sample after pre-amplification, measured on sample (n = 6) diluted to average Cq of reference genes between 32 and 35.5. Right: direct low = measurement (n = 6) on a similar diluted sample without pre-amplification.