Cdna synthesis kit

Isolated RNA was transcribed to complementary DNA (cDNA) using cDNA Synthesis Kit (Biozym) according to manufacturer’s manuals and as described previously^{6 (link),7 (link)}. TaqMan MircoRNA Reverse Transcription Kit (Applied Biosystems) was applied to synthesize cDNA of cel-miR-39-3p working as control. According to the literature^{12 (link),13 (link)} specific primers for circ_0005402, circ_0035560, ANXA2, MBOAT2, TMEM56 and DNAJC6 were validated and afterwards quantitative real-time PCR was performed amplifying the cDNA of the four last mentioned circRNAs using iQSYBR Green Supermix (Bio-Rad). For amplifying cel-miR-39 with quantitative real-time PCR, a specific TagMan MicroRNA assay and ViiA7 machine (Applied Biosystems) was used.
Following primers were used in this study:

circ_0005402 forward	TTTCGGACACATCTGGTGAC
circ_0035560 forward	CACCTGGAGACGGTGATTTT
rev-circ_universal	CCGCTCAGCATCAAAGTTAGT
MBOAT2 forward	AGTGCAAGATAAAGGCCCAAA
MBOAT2 reverse	TGATCATCATAGGAGTGGAGAACA
TMEM56 forward	CATCATTGTGCGTCCCTGTATG
TMEM56 reverse	GCTGAGACTATTGAAACCTGGAGA
DNAJC6 forward	CCAGACATCTTGACCACTACACA
DNAJC6 reverse	ATGTGTCTTTGAGGGTGTCTTT

The Siglec-8 expression on mRNA level in MCF-7, MDA-MB-231 and T-47D BC cell lines was determined using quantitative real-time (RT)- Polymerase chain reaction (PCR). The RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to obtain the total RNA from cultured cells. The RNA was converted to cDNA with a cDNA Synthesis Kit (Biozym 331470L) according to the manufacturer’s instructions. For the RT-PCR, a 20 µL reaction mixture was made up as follows: 1 µL TaqMan^® Gene Expression Assay 20× (Applied Biosystems, Darmstadt, Germany, target GAPDH Nr Hs99999905_m1, target Siglec-8 Nr. Hs00274289_m1, target Gal-7 Nr. Hs00170104_m1, primer sequences are not available due to the use of a commercial assay), 10 µL TaqMan^® Fast Universal PCR Master Mix 2× (Applied Biosystems, Darmstadt, Germany), 1 µL cDNA template and 8 µL RNase free water per sample. RT-PCR was performed on a 96-well plate (Applied Biosystems) covered with an optical adhesive film. A 7500 Fast Real-Time PCR system (Applied Biosystems) was used to run the PCRs. Initially, for the enzyme activation, heating to 95 °C for 20 s was performed, followed by 40 qPCR-cycles of 3 s of denaturation at 95 °C and annealing for 30 s at 60 °C.

For comparability of further analyses, all RNA samples are adjusted to a concentration of 20 ng/µl. cDNA synthesis is then performed with the cDNA synthesis kit—all priming options (Biozym Scientific GmbH). The synthesis is carried out with random hexamer primers and was performed according to the manufacturer’s instructions.