Random 9 mer primers

Total RNA was extracted using TRIzol reagent (Invitrogen, USA). 0.5 μg RNA was reverse-transcribed with PCR diagnosis kits (TianGen, China) and random 9-mer primers (TAKARA, Japan). PCR reaction system contains 50 ng cDNA, 1 μl of each primer and 1×PCR master Mix (TAKARA). PCR cycling conditions were as follows 94 ºC for 5 min, followed by 35 cycles at 94 ºC for 30 s, 56 ºC for 30 s, and 72 ºC for 30 s with a final extension step of 10 min at 72 ºC. The expression levels of β-actin mRNA were measured as the internal control. The sequences of primers were as follows: CLDN6: forward, 5'-TTCATCGGCAACAGCATCGT-3' and reverse, 5'-GGTTATAGAAGTCCCGGATGA-3' (amplification length, 345 bp); β-actin, forward, 5'-TACCTCCCAAGTCCTGTATGAG-3' and reverse, 5'-TGAGCAGCATCAAACTGTGTAG-3' (amplification length, 180 bp). Reactions were carried out in triplicate. The results were analyzed using Quantity One 4.4.1 software (Bio-Rad Laboratories Inc, Hercules, California, USA).

RNA was isolated from frozen specimens using TRIzol reagent (Sangon, Shanghai, China) according to the manufacturer’s instructions. One microgram RNA was reverse-transcribed using M-MLV Kit (TAKARA, Shiga, Japan) and random 9-mer primers (TAKARA). Semiquantitative PCR were conducted using 50 ng of reverse-transcribed cDNA and 0.4 μM of each primer in a final reaction volume of 20 uL containing 1 × PCR master Mix (TAKARA). Typical real-time PCR reactions were performed as previously described [20 (link)]. The relative expression was based on the expression ratio of a target gene versus that of GAPDH. The primers used were as follows: claudin-2 forward primer (5′-CCAACCTCAGCCAGAGAGAGG-3′) and claudin-2 reverse primer (5′-TCCCCAAACCCACTAATCACA-3′); claudin-5 forward (5′-CCTTCATCGGCAACAGCATC-3′) and reverse (5′-CGTACACCTTGCACTGCATC-3′); claudin-8 forward (5′-GTCAGGTCTGTGTTCCATG-3′) and reverse (5′-TGACACCGC CAATGATGC-3′′); claudin-9 forward (5′-ATGGTAGCCACTTGCCTTC-3′) and reverse (5′-TTAGACATGGGCACTCTTGG-3′); and glyceraldehyde phosphate dehydrogenase (GAPDH) forward (5’-AACGTGTCAGTCGTGGACCTG-3′) and reverse (5’-AGTGGGTGTCGCTGTFGAAGT-3′).