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The housekeeping GAPDH (1 2000, Santa Cruz Biotechnology) was detected as a normalized control.
The endogenous actin gene was used as a normalized control gene.
USA300 was used as a normalized control to measure sample expression.
Renilla luciferase acted as a reporter gene and firefly luciferase as a normalized control for each individual analysis.
TaqMan® MicroRNA Assays (Applied Biosystems, CA, USA) was used as the probe for has-miR-195 and has-U6 which act as a normalized control.
β-actin was used as a normalized control, using the following primers: 5′-GCA CCC AGC ACA CAA TGA AG-3′ (forward) and 5′-GCA CCC AGC ACA ATG AAG-3′ (reverse).
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The transfection of SV40- β-gal plasmid was used as the normalized control.
The comparative CT method was used to quantify the expression of SPL, SGPP1 and SGPP2 using GAPDH as the normalized control.
The relative expression level of each candidate gene was calculated using GAPDH as the internal normalized control with the same calibrator.
All results in BC50%CR, 50%CR, BC100%CR and 100%CR for each gene are expressed as a normalized percentage of the control group.
Each mRNA level was determined by qRT-PCR, and presented as a normalized value relative to the control act1 + mRNA (Fig. 2B).
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