FGF10 Cardiac Differentiation Temporal gene expression profiling FGF-13, forward: 59-GAAATCCAATGCCTGCAAGT-39, re- RNA was extracted from ES cells, EBs at various time points verse: (day 2, day 4, day 6 and day 8), ES cell-derived cardiac precursors 59-AGGTGTGAAATGTTCCGAGG-39; and neonatal mouse cardiomyocytes using Trizol reagent FGF-14, forward: 59-AGAGCCTGGTTTTTGGGATT-39, (Invitrogen Carlsbad, CA) according to the manufacturer’s reverse: protocol. Semi-quantitative reverse transcription-polymerase 59-GTTGACTGGTTTGCCTCCAT-39; chain reaction (RT-PCR) was subsequently performed. The FGF-15, forward: 59-GCTGGTCCCTATGTCTCCAA-39, primer sequences used for RT-PCR were as follows: reverse: GAPDH, forward: 59-AACGACCCCTTCATTGAC-39, reverse: 59-TGGTCCTGGAGCTGTTCTCT-39; 59-TCCACGACATACTCAGCAC-39; FGF-16, forward: 59-GTCTTTGCCTCCTTGGACTG-39, Oct4, forward: 5-9CACGAGTGGAAAGCAACTCA-39, re- reverse: verse: 59AGATGGTGGTCTGGCTGAAC-39; 59-TCTCTCCGAGTCCGAGTGTT-39; Brachyury, forward: 59-GCTCATCGGAACAGCTCTCCAA- FGF-17, forward: 59- TCAAACACAGGGGGAGAATC-39, CC-39, reverse: reverse: 59-GGAGAACCAGAAGACGAGGACGTG-39; 59- TCTGCTGCCGAATGTATCTG -39; Flk-1, forward: 59-GCTTTCGGTAGTGGGATGAA-39, reverse: FGF-18, forward: 59-GTGCTTCCAGGTTCAGGTGT-39, 59-GGAATCCATAGGCGAGATCA-39; reverse: Gata4, forward: 59-GCAGCAGCAGTGAAGAGATG-39, reverse: 59-AGCCCACATACCAACCAGAG-39; 59-GCGATGTCTGAGTGACAGGA-39; FGF-21, forward: 59- CTGGGGGTCTACCAAGCATA -39, aMHC, forward: 59-GAGGACCAGGCCAATGAGTA-39, re- reverse: verse: 59- CAGGATCAAAGTGAGGCGAT -39 59-GCTGGGTGTAGGAGAGCTTG-39; FGFR-1b, forward: 59-TTCAGTGGCTGAAGCACATC-39, Cardiac troponin I (cTnI), forward: 59-CTCCTCTGCCAAC- reverse: TACCGAG-39, reverse: 59-CATGCAGAGTGATGGGAGAG-39; 59-CTCAAACTTTTTCTTGCGGC-39; FGFR-1c, forward: 59-TTCAGTGGCTGAAGCACATC-39, Cardiac troponin T (cTnT), forward: 59-ATCCCCGATGGA- reverse: GAGAGAGT-39, reverse: 59-GGAAGTCGCTCTTCTTGGTG-39; 59-TTCCCACGAGTTTTGGAGAC-39; FGFR-2b, forward: 59-CTGCCTGGTGGAGAATGAAT-39, Mlc1a, forward: 59-CCTCAAGGACTCTGCCTTTG-39, reverse: reverse: 59-TCTTCTCTCCCAGGGTAGCA-39; 59-TCACATTGAACAGAGCCAGC-39; Mlc2v, forward: 59-AGCCTTCACAATCATGGACC-39, reverse: FGFR-2c, forward: 59-GGAGGGGATGTGGAGTTTGT-39, 59-CCTCCCTGCTTGTGTGGTCA-39; reverse: FGF-1, forward: 59-GGTTCAACCTGCCTCTAGGA-39, reverse: 59-CAGAACTGTCAACCATGCA-39; 59-ATAAAAGCCCTTCGGTGTCC-39; FGFR-3b, forward: 59-GGAGTTCCACTGCAAGGTGT-39, FGF-2, forward: 59-TATCAAGGGAGTGTGTGCCA-39, reverse: reverse: 59-TATGGCCTTCTGTCCAGGTC-39; 59-ACGCAGAGTGATGGGAAAAC-39; FGF-3, forward: 59-CTTCGGATCACTACAACGCA-39, reverse: FGFR-3c, forward: 59-GGAGTTCCACTGCAAGGTGT-39, 59-GGGCAGGAAGAGAGAGGACT-39; reverse: FGF-4, forward: 59-CGAGGGACAGTCTTCTGGAG-39, re- 59-CCAGCCTCATCAGTTTCCAT-39; verse: FGFR-4, forward: 59-CGTGGTCGTCACTGGTACAA-39, 59-GACACTCGGTTCCCCTTCTT-39; reverse: FGF-5, forward: 59-GTCTTCTGCCTCCTCACCAG-39, reverse: 59-TGATGGAGGTTAAGGAGTC-39. 59-GAAGTGGGTGGAGACGTGTT-39; FGF-6, forward: 59-AATTGGGAAAGCGGCTATTT-39, reverse: Quantitative real-time PCR 59-CCCGTCCATATTTGCTCAGT-39; RNA of treated and non-treated EBs were extracted on day 3 FGF-7, forward: 59-CAAACGGCTACGAGTGTGAA-39, re- using Trizol reagent (Invitrogen) according to the manufacturer’s verse: protocol. RNA was analyzed with quantitative real-time PCR 59-TAAGGCAACGAACATTTCCC-39; (qPCR). Melting curve analyses and PCR product sequencing FGF-8, forward: 59-TTGCACTTGCTGGTTCTCTG -39, were performed to verify primer specificities. qRT-PCR was reverse: repeated at least three times using the following conditions. Each 59- ACTCGGACTCTGCTTCCAAA -39; of the reaction mixtures contained 10 ml of SYBR Green master FGF-9, forward: 59-TTCCCCAACGGTACTATCCA-39, re- mix (Applied Biosystems), 5 pmoles each of forward and reverse verse: primers and 5 ml of 100 times diluted cDNA. The primer 59-CTTTGTCAGGGTCCACTGGT-39; sequences used for qPCR were as follows: FGF-10, forward: 59-TTTTTGGTGTCTTCGTTCCC-39, ndrg1, forward: 59-AGTGCACACGTACCGCCAGC-39, reverse: reverse: 59-ACTGGTGTGCGAGCGGCTTC-39; 59-CTGACCTTGCCGTTCTTCTC-39; gsc, forward: 59 -CCAGCAGTGCTCCTGCGTCC-39, reverse: FGF-11, forward: 59-GTCGCTTTAAGGAGTGCGTC-39, 59-CCCTCTTCTCCGGCGAGGCT-39; reverse: pdgfrb, forward: 59-CACCTTCTCCAGTGTGCTGA-39, reverse: 59-CACTGTGGAGAGAAGGCTCC-39; 59-GGAGTCCATAGGGAGGAAGC-39; FGF-12, forward: 59-TTCCTGTAGGACTGCGTGTG -39, rospo3, forward: 59 -GCTGCCAAGGAGGCTGTGCA-39, reverse: reverse: 59-TTGAGCGTCCTTGCTTTTCT -39; 59-CGGACCCGTGTTTCAGTCCCC-39; PLoS ONE | www.plosone.org 10 December 2010 | Volume 5 | Issue 12 | e14414