Quantitative RT-PCR Protocol for Gene Expression

RNA was isolated using TRI reagent (Sigma) and DNase I-treated (Ambion). cDNA was synthesized using a Maxima first strand cDNA synthesis kit (Fermentas). qRT –PCR was performed using Quantace Sensimix on an Applied Biosystems 7500 machine (Life Technologies Corporation). Primer pairs were previously published (Molkentin et al. 1997 (link); Fujikura et al. 2002 (link); Niwa et al. 2005 (link); Brown et al. 2010a (link)) or designed using Primer3 software. All primers are listed in Supplemental Table S6.

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Wamaitha S.E., del Valle I., Cho L.T., Wei Y., Fogarty N.M., Blakeley P., Sherwood R.I., Ji H, & Niakan K.K. (2015). Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells. Genes & Development, 29(12), 1239-1255.

Publication 2015

TRI reagent DNase I-treated Maxima first strand cDNA synthesis kit Applied Biosystems 7500 machine

Cdna Dnase i Primers Synthesis

Corresponding Organization :

Other organizations : The Francis Crick Institute, Johns Hopkins University, Harvard University

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Variable analysis

independent variables

RNA isolation method (TRI reagent)
CDNA synthesis method (Maxima first strand cDNA synthesis kit)
QRT-PCR method (Quantace Sensimix on Applied Biosystems 7500)
Primer pairs (previously published or designed using Primer3)

dependent variables

Gene expression levels measured by qRT-PCR

control variables

DNase I treatment of RNA samples
Use of positive and negative controls (not explicitly specified)

controls

Positive controls: Not explicitly specified
Negative controls: Not explicitly specified

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