Validation of Differentially Expressed Transcripts

RT-qPCR was used to confirm the lncRNA and circRNA expression profiles obtained from the microarray data. All the samples were normalized to the signal generated by GAPDH (Sangon Biotech, Co., Ltd.) (25 (link)). Data are shown as the fold-change (2^−ΔΔCq) (29 (link)). Student's t-tests were used and P<0.05 was considered to indicate a statistically significant difference. The cDNA was used as the template in an SYBR-Green Real-Time PCR Master Mixes (Invitrogen; Thermo Fisher Scientific, Inc.) and in triplicate subjected to denaturation at 94°C for 4 min and 35 cycles of 94°C for 30 sec, and 60°C for 30 sec, followed by extension at 72°C for 10 min using the specific primers. The primer sequences were as follows: Circ-0025580: F, 5′-CACGAGGGGCTTGTAGGATA-3′; R, 5′-AGGAAACCAAGCCACCAAG-3′. Circ-0024108: F, 5′-AGGCAAGGGATAACTCTTCTAACAC-3′; R, 5′-TTGGCAAATCTGGCGTGTAA-3′. Circ-0025933: F, 5′-GGAATGGAACGACATGCAAA-3′; R, 5′-GACACACATTGTATTTTCACGACAGT-3′. lnc-KLHDC7A-6:2: F, 5′-GGGCGTGAGGTGTGTGTTTA-3′; R, 5′-CGCTTACAAGCAGCAGGTAG-3′. LOC440173: F, 5′-GAGGTACCAAGAGAAGTGCTGATG-3′; R, 5′-GTTAATGCTTTCGGCCAAGATC-3′. EPB41L4A-AS1: F, 5′-GTCATCTATGGAGAGGAAAGGTACAAA-3′; R, 5′-TGTCACCCCAAACCTCAAATG-3′. SMAD5-AS1_3: F, 5′-GTTCTGGTGGTGATGGCATTG-3′; R, 5′-CATCTGGCTCAGGGTGTTCA-3′. GAPDH: F, 5′-TGACTTCAACAGCGACACCCA-3′; R, 5′-CACCCTGTTGCTGTAGCCAAA-3′.