Table 3.

Sequences and information of the RT-qPCR primers used for validation of the final eight biomarkers. fw = forward, rv = reverse, bp = base pairs.

Target	Primer sequences (5’—> 3’)	Source	Tm	Amplicon size
ARF1	–	BioRad (qHsaCED0045157)	60 °C	–
UBC	fw: CTGGTGCTCCGTCTTAGAGG rv: TTTCCCAGCAAAAGATCAACC	Designed with Primer3	60 °C	169 bp
GAPDH	fw: CAGCCTCAAGATCATCAGCA rv: GTCTTCTGGGTGGCAGTGAT	Designed with Primer3	58 °C 60 °C	135 bp
NDRG1	fw: GACCTGGAGATTGAGCGACC rv: CCCAACCACCAACAGAGCA	Designed with Primer3	60 °C 60 °C	75 bp
ISG20	fw: GCCTCCTACACAAGAGCATC rv: CCCTCGCATCTTCCACCG	Designed with Primer3	61 °C 60 °C	64 bp
FOXM1	fw: AGCGACAGGTTAAGGTTGAGG rv: TCTCAGTGCTGTTGATGGCG	Designed with Primer3	60 °C 61 °C	120 bp
TPX2	fw: ATGATGCCCCCTCGGATTTC rv: AGCCCTCCAGTTCCATTCTTC	Designed with Primer3	60 °C 60 °C	133 bp
MYBL2	fw: CCTGCCTTACAAGTGGGTGG rv: ACCAAGCATCAGGGTCCGA	Designed with Primer3	61 °C 61 °C	107 bp
AHNAK2	fw: GGAAGCGAGATTCAGGGGAC rv: GGACAGCCTCTGGACGACT	Designed with Primer3	60 °C 61 °C	60 bp
GPRC5C	fw: GATGCACAAAGTTCCGTCCG rv: AGTACATGTCTTCAGCCCGC	Designed with Primer3	60 °C 60 °C	113 bp
THBD	fw: GGGGTGATTAGAGGGAGGAGA rv: TTGCCCAGTGGTCCAGTGA	Designed with Primer3	60 °C 61 °C	65 bp

ARF1, UBC and GAPDH were chosen as reference genes for the cell-free RNA, and UBC and GAPDH for the cellular RNA after analyzing Cq values of multiple reference gene candidates from a preliminary Cal62 vandetanib treatment EV experiment with the algorithms NormFinder (https://www.moma.dk/software/normfinder, RRID:SCR_003387)⁹⁹ and geNorm (https://genorm.cmgg.be/, RRID:SCR_006763)¹⁰⁰. Log2FCs were calculated by normalizing the Cq values to the mean Cq of the reference genes and with respect to the PCR amplification efficiency¹⁰¹.