Total RNA from GC cells was extracted using RNeasy Mini Kit (cat. no. 74104; Qiagen GmbH) and then reverse transcribed into cDNA using PrimeScript RT reagent kit (cat. no. RR037A; Takara Bio, Inc.) according to the manufacturer's protocol. RT-qPCR was performed using Premix Ex Taq (cat. no. RR420A; Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min; annealing and elongation at 95°C for 30 sec, 95°C for 5 sec, 60°C for 34 sec for 40 cycles; and final extension at 60°C for 10 min. The sequences of the primer pairs used were: Oct4 forward, 5′-GTGGAGGAAGCTGACAACAA-3′, reverse, 5′-AACAAATTCTCCAGGTTGCC-3′ and probe, 5′-Fam-TCTCTTTCGGGCCTGCACGA-3′Tamra; Sox2 forward, 5′-AATGCCTTCATGGTGTGG−3′, reverse, 5′-CTTCTCCGTCTCCGACAAA-3′, and probe, 5′Fam-AGTTTCCACTCGGCGCCCAG-3′Tamra; KLF4 forward, 5′-GGCACTACCGTAAACACACG-3′, reverse, 5′-CTGGCAGTGTGGGTCATATC-3′, and probe, 5′Fam-CAGGTCGGACCACCTCGCCT-3′Tamra; CD44 forward, 5′-TGGCAACAGATGGCATGAGG-3′, reverse, 5′-CCTTGCATTGGATGGCTGGT-3′ and probe, 5′Fam-AACAGGGACAGCTGCAGCCTCAGCT-3′Tamra; ALDH1 forward, 5′-GCTGGCGACAATGGAGTCAA-3′, reverse, 5′-TGTACGGCCCTGGATCTTGT-3′ and probe, 5′Fam-ACATTGCGCTACTGTGCAGGTTGGGCT−3′ Tamra; Slug forward, 5′-TCACTGTGTGGACTACCGCT-3′, reverse, 5′-TCGCCCCAAAGATGAGGAGT-3′ and probe, 5′Fam-ATTCCACGCCCAGCTACCCAATGGCCT−3′Tamra; Twist forward, 5′-CCCTCGGACAAGCTGAGCAA-3′, reverse, 5′-TCGCTCTGGAGGACCTGGTA-3′ and probe, 5′Fam-ATTCAGACCCTCAAGCTGGCGGCCA-3′Tamra; vimentin forward, 5′-CAATGAGTCCCTGGAACGCC-3′, reverse, 5′-GGTGACGAGCCATTTCCTCC-3′ and probe, 5′Fam-ACCAAGACACTATTGGCCGCCTGCAGG−3′Tamra; GAPDH forward, 5′-ATCATCCCTGCCTCTACTGG-3′, reverse, 5′-GTCAGGTCCACCACTGACAC-3′ and probe, 5′Fam-ACCTTGCCCACAGCCTTGGC-3′Tamra; The relative mRNA expression levels were calculated using 2−ΔΔCq method (21).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.