Protocol detail

Quantitative Real-Time PCR Protocol

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Quantitative Real Time PCR qRT-PCR was performed as described [16 (link)]. Briefly, cDNA was generated from 1μg total RNA using oligo-dT primers and Superscript III polymerase (Invitrogen). Real-time PCR analysis was done with iQ SYBR Green Supermix (Bio-Rad) in the Bio-Rad iCycler for 40 cycles. Primer pairs forward (F) and reverse (R): GAPDH F-ATGACAATGAATACGGCTACAG, R-TCTCTTGCTCAGTGTCCTTG; Mylk F-CCAAGGACCGGATGAAGAAATA, R-CCCTGAGATCATTGCCATAGAG; Sema3d F-TGGGACATAGAAGCATTAG, R-AGAGGCTTGTTGGGATTTAGG; Sxc F-AGGGCCTATGAACAGAGAGAT, R-GTAGAGAGCCAGCATGGAAAG; Cadm1 F-TCTGTAGGCGGCTCAGTATAG, R-CTCACATGTCGGGTCTGTTTAG; Krt8 F-GGCCAACCTTAGGAGGAATTT, R-GAGCCAGCTGAGGCTTTATT; Chst7 F-GTGAGACACTGGGACTGATTTG, R-GCCAAGGTGTCTGTCATTACTT; Versican F-CAGGCTATCACAGGCAGATTAG, R-CAGAAGCCAAGGAGTCATTCA.

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DeLaughter D.M., Clark C.R., Christodoulou D.C., Seidman C.E., Baldwin H.S., Seidman J.G, & Barnett J.V. (2016). Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFβR3 In Vitro. PLoS ONE, 11(8), e0159710.

Publication 2016

Superscript III polymerase iQ SYBR Green Supermix

Cadm1 Cdna Gapdh Krt8 Mylk Oligo dt Primer Real time pcr analysis Sema3d Sybr green Versican

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of GAPDH, Mylk, Sema3d, Sxc, Cadm1, Krt8, Chst7, and Versican genes

control variables

None explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the protocol.

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