Fig. 2

Detection of ZFAS1 isoforms by RT-PCR and demonstration of their subcellular location. a Orientation of primers used in RT-PCR in relation to ZFAS1 genomic arrangement. Primers were designed to cover all isoforms. b Primer pairs used to detect ZFAS1 isoforms and the expected PCR product sizes for different isoforms. c Localisation of ZFAS1 and ZNFX1 in cellular compartments as detected by PCR in MDA-MB-468 cells. ZFAS1 was amplified using primer set E1F3-E5R1 and shown to be present in cytoplasm and nucleus. ZNFX1 was expressed predominantly in the nucleus. NEAT1, a nuclear lncRNA was used as a nuclear control; GAPDH was used as a positive control. d Expression of ZFAS1 isoforms using different primer pairs in MDA-MB-468 cellular fractions. i) E1F3-E5R1 amplified all isoforms except var1, allowing detection of var2-5. (ii) E1F3-E2R2 amplified the first exons of var2-5 in extracts of both cytoplasm and nucleus. (iii) To identify var1, PCR was performed in the first and second exons using primer set E1F1-E2R2, and (iv) verified by internal PCR using primers E1F2-E2R1, yielding a 93 bp product in both cytoplasm and nucleus