Revertra acer

Total RNA was extracted by Sepasol RNA I Super G (Nacalai Tesque) and cDNA synthesized using ReverTra AceR quantitative polymerase chain reaction (qPCR) reverse transcriptase (RT) Master Mix (TOYOBO). qPCR was performed with the Thermal Cycler Dice Real Time System II (Takara Bio) according to the manufacturer’s instructions. The primer sequences for each gene were as follows: EAP1 (forward, 5′-tcgcttcaagaaggaccact-3′ and reverse, 5′-ccgtggggtactcaatgaac-3′); PSA (forward, 5′-ggcagcattgaaccagaggag-3′ and reverse, 5′-gcatgaacttggtcaccttctg-3′); and KLK2 (forward, 5′-tcatccagtctcggattgtg-3′ and reverse, 5′-cttctttaggcaatgggcag-3′); and Nkx3.1 (forward, 5′-gccaagaacctcaagctcac-3′ and reverse, 5′-agaaggcctcctctttcagg-3′); and Rplp0 (forward, 5′-tcgacaatggcagcatctac-3′ and reverse, 5′-tgatgcaacagttgggtagc-3′). RNA levels were normalized using the Rplp0 gene as an internal control.

IEC-6 cells were cultured in DMEM containing 10% FBS until they reached 50–55%
confluence. When the cultured cells reached 50–55% confluence, medium was replaced with
DMEM supplemented with 0.5% FBS containing TGF-β1 (10
ng/ml⁻¹) for 7 days to induce EMT cells.
The medium was changed every 2 days. Total RNA was extracted by using Trizol reagent
(Invitrogen, Tokyo, Japan). First-strand cDNA was synthesized by using a random nine-mer
primer and ReverTra Ace^(R) (a high efficient M-MLV; Moloney Murine Leukemia
Virus reverse transcriptase) (Toyobo, Tokyo Japan) at 30°C for 10 min, 42°C for 1 hr, 99°C
for 5 min, and 4°C for 5 min. PCR amplification was performed by using ExTaq DNA
polymerase. Primers used for PCR analysis are shown in Table 1Table 1.

Primer sets	Orientation	Sequence (5′ to 3′)	PCR product (bp)
Rat-α-SMA (NM_031004)	Forward	GGGAGTGATGGTTGGAATGG	197
	Reverse	CCGTTAGCAAGGTCGGATG
Rat E-cadherin	Forward	ATCTAAAGCTTCACAAGCTGGA	502
	Reverse	TGATCTGTGACTGTGACCACTA
Rat-TnC (XM_008763758.2)	Forward	ATGTTGAATGGCGACAC	188
	Reverse	CGGTCTCCAAACCCAG
Rat-GAPDH (XM_576394)	Forward	TCCCTCAAGATTGTCAGCAA	308
	Reverse	AGATCCACAACGGATACATT

. After an initial check, we selected 32 cycles for α-SMA, E-cadherin, and
tenascin-C.