RNA Extraction and qPCR Analysis

RNA was extracted using trizol and analyzed by SYBRgreen qPCR with the 7300 real time PCR system (Applied Biosystems)^{67 (link)} and TaqMan qPCR (Applied Biosystems). Primers used are listed in Suppl.Table.2.
qPCR of miRNAs was conducted using TaqMan miRNA assays (Applied Biosystems). Raw Ct values for miRNAs were normalized to RNU66. Linear expression values for all qPCR experiments were calculated using the 2(−ΔCt) method. P-values were calculated using a student's t-test.

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Sperber H., Mathieu J., Wang Y., Ferreccio A., Hesson J., Xu Z., Fischer K.A., Devi A., Detraux D., Gu H., Battle S.L., Showalter M., Valensisi C., Bielas J.H., Ericson N.G., Margaretha L., Robitaille A.M., Margineantu D., Fiehn O., Hockenbery D., Blau C.A., Raftery D., Margolin A., Hawkins R.D., Moon R.T., Ware C.B, & Ruohola-Baker H. (2015). The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition. Nature cell biology, 17(12), 1523-1535.

Publication 2015

7300 real time PCR system TaqMan qPCR TaqMan miRNA assays

Assays Mirnas Primers Trizol

Corresponding Organization :

Other organizations : University of Washington, University of Portland, University of California, Davis, Fred Hutch Cancer Center, University of Washington Medical Center

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Variable analysis

independent variables

RNA extraction method (Trizol)
QPCR method (SYBRgreen, TaqMan)

dependent variables

MRNA expression levels
MiRNA expression levels

control variables

Applied Biosystems 7300 real-time PCR system
RNU66 for miRNA normalization

controls

No positive or negative controls were explicitly mentioned.

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