MCF-7 cell growth, transfection and inhibition of the nonsense-mediated decay were performed as previously described [16]. The triple-negative breast cancer (TNBC) cell line MDA-MB-231 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids and 1% penicillin/streptomycin solution.

RNA was purified using the Genematrix Universal RNA Purification Kit (EURx, Gdansk, Poland), with on-column DNAse I digestion. RNA (400 ng) was retrotranscribed using the vector exon V2-specific primer RTPSPL3-RV (5′-TGAGGAGTGAATTGGTCGAA-3′) and the RevertAid First-Strand cDNA Synthesis Kit (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. Two μL of cDNA were used for amplification of the regions of interest using Platinum Taq polymerase (Life Technologies). To increase specificity, cDNA was amplified, with one primer located in one RAD51C exon of the insert and another one in a vector exon (V1 or V2). Thus, for variants of the donor site of exon 2, the amplification was performed using primers SD6-PSPL3_RTFW (5′-TCACCTGGACAACCTCAAAG-3′) and RTR51D_9-rv (5′-GTCCCTGTCTCGAGTTATG-3′) (amplicon size = 843 bp). For the remaining variants (exons 3 to 8), the following primers were used: RTR51D_ex2-fw (5′-TAGCTCAGAAATGTGGCTT-3′) and RTpSAD-RV (Patent P201231427, CSIC) (size = 885 bp). Samples were denatured at 94 °C for 2 min, followed by 35 cycles × (94 °C, 30 s/59 °C, 30 s/72 °C, 1 min/kb) and 72 °C for 5 min. RT-PCR products were sequenced by Macrogen (Madrid, Spain).

To estimate the relative proportions of each transcript, semi-quantitative fluorescent RT-PCRs were performed using a FAM-labeled primer under standard conditions, except that 26 cycles were herein applied [14,27,28]. Fluorescent products were run with LIZ-1200 Size Standard by Macrogen (Seoul, Korea) and analyzed using Peak Scanner software V1.0 (Life Technologies). The mean peak areas of three independent experiments of each variant were used to calculate the relative proportions of each transcript as well as the standard deviations.

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