Abi 7300 sequence detection system

For the RT-qPCR analyses of miR-338-3p expressions, total RNA of tissues and cells was extracted using TRIzol^® RNA reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and first-strand cDNA was prepared via RT with Superscript II reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The RT-PCR sample was incubated at 42°C for 50 min and heated to inactivate the enzyme at 70°C for 15 min. The expression of miR-338-3p was quantified using Fast SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) under ABI 7300 Sequence Detection System (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol and normalized to the expression of U6. The primers were as follows: Forward (F)-miR-338-3p (5′-TGCGGTCCAGCATCAGTGAT-3′) and reverse (R)-miR-338-3p (5′-CCAGTGCAGGGTCCGAGGT-3′), F-U6 (5′-TGCGGGTGCTCGCTTCGGCAGC-3′) and R-U6 (5′-CCAGTGCAGGGTCCGAGGT-3′). The PCR temperature protocol was set as follows: The sample was incubated for 30 min at 16°C, followed by 60 cycles at 30°C for 30 sec, 42°C for 30 sec and 50°C for 1 sec, and incubated at 85°C for 5 min to inactivate the reverse transcriptase. The relative expression of each gene was calculated and normalized by the 2^−ΔΔCq method (18 (link)). Each sample was tested in triplicate.