Sybr green dye

Total RNA was extracted from tissues or cultured cells using TRIzol reagent (Invitrogen, USA) as described in the protocols. After reverse transcription using cDNA reverse transcriptase (Genecopoeia, Rockville, MD) and Oligo(dT) primers, the mRNA levels of ANXA2, GPC1, and β-actin were analyzed by RT-qPCR using SYBR Green dye (Genecopoeia, Rockville, MD) and Quant Studio 5 RT-qPCR instrument (Applied Biosystems, USA). The mRNAs for ANXA2 and GPC1 were amplified and quantified with the primers listed below. The synthetic oligonucleotide primer sequences for AnnexinA2, Glypican1 and β-actin were as follows: h-AnnexinA2 5′-GATCAGAATCATGGTCTCCCG-3′ (upstream) and 5′-GCCCTTAGTGTCTTGCTGGAT-3′ (downstream); h-Glypican1 5′-TGACTATTGCCGAAATGTGCT-3′ (upstream) and 5′-TCCTGGAGGGCGTTGATG-3′ (downstream); h-β-actin 5′-TAGTTGCGTTACACCCTTTCTTG-3′ (upstream) and 5′-TCACCTTCACCGTTCCAGTTT-3′ (downstream). Amplification conditions were set as follows: predenaturation at 95 °C for 15 min, 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 60 s. AnnexinA2 and Glypican1 mRNA levels were normalized to that of β-actin.

Total RNA isolated by TRIzol reagent (Invitrogen, USA) was converted to complementary DNA (cDNA) by a cDNA Synthesis Kit (Gene Copoeia, USA) as per the manufacturer’s instructions. Subsequently, the cDNAs were amplified by real-time qPCR with specific primers (Supplementary Tab. 1) and SYBR-Green dye (Gene Copoeia, USA). PCRs were performed with the following parameters: pre-denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, extension at 72 °C for 30 s. β-Actin was used as an internal control.